BIOLOGY 

LIBRA** 

G 


A   LABORATORY   COURSE   IN   SERUM   STUDY 


THE  MACMILLAN  COMPANY 

NEW  YORK  •    BOSTON  •   CHICAGO  •   DALLAS 
ATLANTA  •    SAN   FRANCISCO 

MACMILLAN  &  CO.,  LIMITED 

LONDON  •    BOMBAY  •    CALCUTTA 
MELBOURNE 

THE  MACMILLAN  CO.  OF  CANADA,  LTD. 

TORONTO 


A   LABORATORY    COURSE 
IN  SERUM  STUDY 

BACTERIOLOGY  208 


BEING  A  SERIES   OF  EXPERIMENTS   AND   DIAGNOSTIC 
TESTS    IN     IMMUNOLOGY    CARRIED    OUT    IN    AN 
OPTIONAL   COURSE   GIVEN   TO  MEDICAL  AND 
GRADUATE    STUDENTS   IN   THE    DEPART- 
MENT   OF   BACTERIOLOGY,    COLLEGE 
OF    PHYSICIANS   AND   SURGEONS 
COLUMBIA     UNIVERSITY 
NEW  YORK,  BY  THE 
WRITERS 


HANS   ZINSSER,   M.D. 

\\ 

J.    G.    HOPKINS,   M.D. 
REUBEN   OTTENBERG,   M.D. 


SECOND  EDITION  REVISED 


THE    MACMILLAN    COMPANY 
1921 

AH  rights  reserved 


-   -     .     *..> 


- 

. 


a 


COPYRIGHT,  1916,  1921, 
BT  THE  MACMILLAN  COMPANY. 


Sot  up  and  f  lectrotyped.    Published  February,  1916. 
New  Edition  Revised.     Published  January,  1921. 


PREFACE  TO  SECOND  EDITION 

THE  present  issue  of  the  Laboratory  Course  in  Serum  Study 
does  not  represent  a  new  edition  in  that  it  has  been  found  impos- 
sible to  completely  revise  the  book  at  this  time.  There  are,  how- 
ever, a  number  of  important  changes  in  protocols  and  technique 
which  have  been  made  on  the  basis  of  experience  hi  giving  the 
course  to  medical  and  public  health  students.  Most  of  these 
changes  are  in  the  form  of  alterations  hi  the  plan  of  experiments, 
making  them  more  easy  to  perform  and  more  illustrative  of  under- 
lying principles.  In  preparing  these  changes  for  the  new  printing, 
we  have  been  greatly  aided  by  Dr.  Samuel  Cochran,  Professor  of 
Bacteriology  at  the  Shantung  Christian  University  hi  China,  who 
helped  us  hi  giving  this  course  at  Columbia  last  year. 


iva 


INTRODUCTION 

THE  course  here  outlined  is  given  by  the  authors  at  Colum- 
bia University.  The  prerequisite  theoretical  knowledge  is  pre- 
sented in  a  series  of  lectures  based  on  the  textbook  "  Infection 
and  Resistance/'  by  the  senior  author. 

Immunity,  like  other  branches  of  science,  cannot  be  taught 
without  experiment  and  demonstration.  For  this  reason  we 
have,  for  several  years,  supplemented  our  lecture  course  on 
Infection  and  Resistance  by  an  optional  course  on  Serum  Tech- 
nique. Our  purpose  in  this  has  been  not  so  much  to  teach 
beginners  to  carry  out  practical  diagnostic  tests  as  to  allow  the 
student  to  carry  out  fundamental  experiments,  and,  in  drawing 
conclusions  from  his  results,  to  learn  to  reason  from  protocols 
and  in  this  way  discover  the  basic  principles  for  himself. 

It  has  been  our  contention  for  a  number  of  years  that 
thorough  instruction  in  the  phenomena  of  immunity  consti- 
tuted a  logically  necessary  preparation  for  the  clinic  on  infec- 
tious diseases.  For  this  reason  our  courses  have  been  offered 
as  optionals  to  second  and  third  year  medical  students.  Con- 
trary to  ordinary  belief,  students  at  this  stage  of  preparation 
have  found  no  difficulty  in  comprehending  the  work,  and  have, 
we  think,  derived  benefits  in  experimental  methods  and  reason- 
ing far  beyond  the  actual  gain  in  new  facts.  Though  optional 
now,  these  courses  we  hope  may  eventually  become  integral, 
required  parts  of  the  regular  medical  curriculum  —  the  lectures 
and  demonstrations  correlated  with  —  the  laboratory  course  fol- 
lowing —  the  course  in  Bacteriology.  This,  however,  we  realize 
may  have  to  await  the  lengthening  of  the  medical  course  as 
a  whole.  Meanwhile  such  a  course  can  certainly  be  optionally 


Vi  INTRODUCTION 

available  for  students  who  have  the  desire  to  take  it  —  and 
our  experience  at  two  medical  schools  has  taught  us  that  there 
are  always  a  good  many  who  do. 

The  little  volume  has  been  compiled  in  the  first  place  for 
our  own  convenience  from  the  protocols  given  to  our  students. 
If  incidentally  it  is  of  service  to  others  in  planning  similar 
courses,  or  to  laboratory  workers  in  repeating  experiments, 
we  shall  be  pleased.  The  book  as  it  stands  is,  of  course,  not 
a  manual  of  immunity.  The  course  should  follow  or  be  syn- 
chronous with  lectures,  reading,  and  demonstrations  on  the 
principles  of  infection  and  immunity.  In  our  own  work  the 
course  is  offered  to  students  who  have  had  work  in  general 
Bacteriology  and  have  taken  the  lecture  course  on  Infection 
and  Resistance,  and  reading  is  assigned  in  the  textbook  on 
this  subject. 

Since  the  manual  has  grown  out  of  the  course  after  some 
four  years  of  experimentation  and  adaptation  to  classroom  pos- 
sibilities, it  in  no  way  represents  an  "  ideal "  formula  but  de- 
scribes only  work  actually  done  with  students.  For  this  reason 
we  have  often  simplified  the  experiments  in  a  way  which  would 
be  undesirable  in  actual  research  work.  But  we  have  not  done 
this  at  the  expense  of  exactitude.  Thus  we  have  in  many 
cases  adapted  the  technique  to  the  possibilities  of  a  single  after- 
noon with  a  class  not  yet  fully  trained.  In  order  to  facilitate 
the  giving  of  such  a  course  by  other  teachers  we  have  added 
a  time  schedule  of  the  whole  course,  and  have  given  in  each 
exercise  a  list  of  the  materials  needed. 


TIME   SCHEDULE   OF  LESSONS 


SUBJECT  OF  LESSON 


I.    IMMUNIZATION  OF  ANIMALS. 


II.     BACTERICIDAL  AND  H.EMO- 
LYTIC  POWER  OF  NORMAL 
SERUM. 
III.     H^MOLYSIS. 


IV.    QUANTITATIVE    RELATIONS 
OF      AMBOCEPTOR      AND 
COMPLEMENT. 
V.     QUANTITATIVE    RELATIONS 

(Continued). 

VI.     REVERSIBILITY  AND  SPEED 
OF    UNION    OF    IMMUNE 
BODY  AND  CELLS. 
VII.     PFEIFFER  PHENOMENON. 


WORK  PRELIMINARY  TO  SUBSE- 
QUENT LESSONS 

Preparation  of  B.  typhosus. 

B.  coli. 

Staphylococcus     au- 
reus. 

Vibrio  choleras. 

Sheep  serum. 

Ascitic  serum. 

Sheep  red  cells. 

Cat  red  cells. 

Horse  serum. 

Human  serum. 
Injection  of  rabbits. 
Repetition  of  injections  of  Lesson  I. 


Repeat  injections  of  Lesson  I. 

Commence  collection  of  guinea  pig 
hearts  in  absolute  alcohol  for 
Wassermann  antigen  (Lesson 
XIX). 

Repeat  injections  of  Lesson  I. 


Repeat  certain  injections  of  Les- 
son I. 

Trial  bleeding  of  animals  which  re- 
ceived their  last  injections  at  4th 
session. 

Bleeding  of  animals  of  Lesson  I. 


1  Arranged  for  two  afternoons  a  week  for  a  term  of  three  and  a  half  months. 

vii 


Vlll 


TIME    SCHEDULE    OF   LESSONS 


SUBJECT  OF  LESSON 

VIII.     BACTERICIDAL    TESTS    IN 
VITRO. 

IX.     AGGLUTININS. 


X.  AGGLUTINATION — MICRO- 
SCOPIC METHOD.  EF- 
FECT OF  SALT. 

XI.     ABSORPTION  OF  AGGLUTI- 
NINS. 

XII.  IN  Vivo  EFFECT  6r  H^EM- 
AGGLUTINATIVE  AND 
H^MOLYTIC  SERA. 

XIII.  ISOH^MOLYSIS    AND    IsO- 

AGGLUTINATION. 

XIV.  PRECIPITINS. 


XV.    FORENSIC 
TEST. 


PRECIPITIN 


XVI.     BORDET-GENGOU  PHENOM- 
ENON. 


XVII.    ALEXIN  FIXATION  BY  SPE- 
CIFIC PRECIPITATES. 

XVIII.    FORENSIC      COMPLEMENT 
FIXATION. 

XIX.  WASSERMANN  ANTIGEN  : 
PREPARATION  AND  Ti- 

TRATION    OF    ANTIGEN. 

XX.    WASSERMANN  REACTION  : 
TECHNIQUE  OF  TESTS. 

XXI.  COMPLEMENT  FIXATION 
WITH  BACTERIAL  EX- 
TRACTS. 


WORK  PRELIMINARY  TO  SUBSE- 
QUENT LESSONS 

Titrate  hemolytic  sera  made  by 
class. 

Inoculate  flasks  with  virulent  diph- 
theria strain  for  production  of 
diphtheria  toxin  (Lesson  XXII). 


Filter    diphtheria     toxin    (Lesson 
XXII). 


Begin  preparation  of  Wassermann 
antigens  for  Lesson  XIX. 

Inject  guinea  pigs  with  diphtheria 
toxin  for  determination  of  mini- 
mal lethal  dose  (Lesson  XXII). 

Finish  preparation  of  simple  alco- 
holic Wassermann  antigen  and 
Noguchi  antigen. 

Inject  guinea  pigs  with  diphtheria 
toxin-antitoxin  mixtures  for  de- 
termination of  L  dose  (Lesson 
XXII). 


TIME    SCHEDULE   OF   LESSONS 


IX 


SUBJECT  OF  LESSON 
XXII.     DIPHTHERIA  TOXIN  :  Ti- 

»  TRATION     OF     AN    UN- 

KNOWN ANTITOXIN. 

XXIII.  TETANUS   TOXIN;    TET- 

ANOLYSIN  AND  ANTI- 
TETANOLYSIN  J  TET~ 
ANOSPASMIN. 

XXIV.  COBRA    VENOM,    KICIN, 

SAPONIN. 

XXV.     ANTITRYPSIN. 
XXVI.    NORMAL  OPSONINS. 
XXVII.     OPSONIC        INDEX: 
WRIGHT'S  TECHNIQUE. 
XXVIII.     HEAT      STABILITY      OF 
OPSONINS. 

XXIX.       TlTRATION    OF    XORMAL 

OPSONINS  BY  DILU- 
TION METHOD. 

XXX.  TlTRATION  OF  IMMUNE 
OPSONINS  BY  DILU- 
TION METHOD. 

XXXI.     PREPARATION    OF    BAC- 
TERIAL VACCINES. 
XXXII.     ANAPHYLAXIS. 


WORK  PRELIMINARY  TO  SUBSE- 
QUENT LESSONS 


Inject  guinea  pigs  with  serum  for 
Lesson  XXXII. 


TO  BE  PREPARED  OR  OBTAINED  BY  INSTRUCTOR 


BEFORE  COURSE. 


BEFORE  LESSON  I. 


BEFORE  LESSON  IL 


BEFORE  LESSONS  III,  IV,  V,  AND 
VI. 

BEFORE  LESSON  VII. 


BEFORE  LESSON  VIIL 


BEFORE  LESSON  IX. 

BEFORE  LESSON  X. 
BEFORE  LESSON  XI. 

BEFORE  LESSON  XII. 


Haemolytic  (antisheep)  serum. 

Immunize  a  guinea  pig  to  cholera 
spirillum. 

Inoculate  students  against  typhoid. 

One  day  before  lesson  plant  agar 
slants  of  B.  typhosus,  B.  coli,  Sta- 
phylococcus  aureus,  Vibrio  cho- 
lerae. 

Obtain  sheep  serum,  ascitic  fluid, 
horse  serum,  sheep's  blood,  two 
cats,  ten  rabbits,  one  guinea  pig. 

One  day  before,  inoculate  broth  cul- 
ture of  B.  typhosus. 

Fresh  dog  serum. 

Washed  guinea  pig  cells. 

Washed  human  cells. 

Sheep's  blood  (washed). 

Fresh  guinea  pig  serum. 

Diluted  haemolytic  serum. 

Plant  culture  of  virulent  cholera 
vibrios. 

Guinea  pig  immunized  to  cholera. 

24  hours  before,  plant  broth  culture 
of  B.  typhosus. 

Obtain  sterile  blood  from  normal 
rabbit. 

24  hour  broth  culture  typhoid. 

Virulent  diphtheria  strain. 

24  hour  broth  culture  B.  typhosus. 

Slant  cultures  of  B.  typhosus  and 
B.  coli. 

Two  cats,  one  rabbit. 


Xll 


TO   BE    PREPARED   OR   OBTAINED   BY   INSTRUCTOR 


BEFORE  LESSON  XIII. 

BEFORE  LESSON  XV 
BEFORE  LESSON  XVI. 


BEFORE  LESSON  XVIII. 
BEFORE  LESSON  XIX. 
BEFORE  LESSON  XXIII. 


BEFORE  LESSON  XXIV. 


BEFORE  LESSON  XXV. 


BEFORE  LESSON  XXVI. 


BEFORE  LESSON  XXVII. 


BEFORE  LESSON  XXVIII. 


BEFORE  LESSON  XXIX. 


Beef  hearts. 

Absolute  alcohol  for  Wassermann 
antigens. 

Prepare  blood  stains  to  be  tested. 

Typhoid  slants. 

Obtain  standard  diphtheria  anti- 
toxin. 

Horse    meat.      Give    to    class    at 
previous  exercise. 

Syphilitic  and  normal  human  sera. 

Tetanus  toxin  and  antitoxin.     Test 

haemolytic  power  of  toxin. 
Guinea  pig  brains. 
Guinea  pigs. 

Cobra  venom.     Saponin,  ricin,  leci- 
thin. 
Guinea  pig's  blood.     Sheep  blood. 

Prepare  alkaline  casein  solution  and 

trypsin  solution. 
Obtain  blood  from  carcinoma  and 

other  patients. 

Inject  guinea  pigs  with  aleuronat. 
Slants  of  Staphylococcus  aureus. 
Guinea  pig  serum. 

Inoculate  slants  with  Staphylococ- 
cus aureus. 

Glass  tubing  for  making  Wright's 
capsules  and  pipettes. 

Rubber  nipples. 

Triangular  files. 

Citrate  solution. 

Staphylococcus  immune  guinea  pig 

serum. 

Staphylococcus  emulsions. 
Guinea  pigs  for  preliminary  work 

for  Lesson  XXXII. 
Horse  serum. 
Sheep  serum. 

Guinea  pig  serum. 
Same    materials    as    for    Lessons 
XXVII  and  XXVIII. 


TO  BE  PREPARED  OR  OBTAINED  BY  INSTRUCTOR 


Xlll 


BEFORE  LESSON  XXX. 


BEFORE  LESSON  XXXI. 


BEFORE  LESSON  XXXII. 
BEFORE  LESSON  XXXIII. 


Serum  of  rabbit  immunized  against 

staphylococcus. 

Inject  guinea  pig  with  aleuronat.     * 
Serum  of  normal  rabbit. 
Other  materials  same  as  for  three 

preceding  lessons. 
Agar  slants  of  Staphylococcus  au- 

reus. 

Sterile  tubes  with  glass  beads. 
Hsemocytometers. 
Methylene  blue  solution. 
Wright's  stain  or  Jenner's  stain. 
Hopkins  centrifuge  tube. 
Horse  serum. 
Sheep  serum. 
Immune  anti-horse  serum  injected 

into  guinea  pigs  day  before  lesson. 
Horse  serum. 


A   LABORATORY   COURSE   IN   SERUM   STUDY 


2  LABORATORY  COURSE  IN  SERUM  STUDY 

LESSON  I 

STUDENTS  in  this  course  will  prepare  their  own  materials  for  injec- 
tion, will  immunize  and  bleed  animals  themselves,  in  fact  will  do  all  the 
manipulations  necessary  in  the  preparation  of  materials  for  the  experi- 
ments that  are  done  in  the  class.  In  some  cases  students  will  work  in 
groups  of  two  or  more  in  order  to  save  time  and  animals. 

It  is  suggested  that  each  student  be  required  to  enter  his  results  on  a 
joint  tabulation  sheet  prepared  for  each  day's  exercise  and  that  these  results 
be  discussed  by  the  instructor  at  the  beginning  of  the  following  exercise. 

PREPARATION  OF  MATERIALS  FOR  INJECTION 

1.  SHEEP  ERYTHROCYTES 

The  students  who  are  to  immunize  their  rabbit  to  blood  cells 
should  obtain  the  blood  from  the  sheep,  and  at  subsequent  lessons 
other  students  will  be  given  experience  in  this  procedure. 

Bleeding  of  Sheep.  —  The  animal  is  held  by  two  assistants 
with  the  head  thrown  back  and  one  side  of  the  neck  is  clipped 
and  carefully  shaved. 

A  rubber  tourniquet  is  then  placed  around  the  neck  as  near 
as  possible  to  the  shoulders  and  tied  tightly  so  as  to  compress  the 
external  jugular  vein.  The  vein  distends  and  will  be  seen  to  stand 
out  as  a  ridge  the  size  of  a  finger.  A  small  part  of  the  shaved  area 
of  the  skin  is  painted  with  tincture  of  iodine,  and  a  large  sterile 
needle  with  four  inches  of  rubber  tubing  attached  is  plunged 
through  the  skin  into  the  vein.  The  blood  is  allowed  to  flow 
into  small  Erlenmeyer  flasks  containing  about  20  large  beads, 
and  when  the  desired  amount  of  blood  is  obtained  the  flask  is 
shaken  thoroughly  until  coagulation  is  completed,  this  procedure 
defibrinating  the  blood.  If  preferred,  the  blood  may  be  received 
into  citrate  solution,  containing  1  per  cent  sodium  citrate  and  0.5 
per  cent  sodium  chloride  (not  more  than  1  part  of  blood  to  1  part 
of  solution)  or  into  ammonium  oxalate  solution  (1  per  cent). 

To  obtain  washed  erythrocytes  and  serum  for  injection 
the  defibrinated  blood  is  poured  into  sterile  centrifuge  tubes. 
The  cotton  plugs  of  these  tubes  should  be  fastened  by  turning 
the  free  edges  back  over  the  neck  of  the  tube  and  securing 
them  there  by  means  of  rubber  bands;  this  will  prevent  the 


4  LABORATORY  COURSE  IN  SERUM  STUDY 

plug  being  thrown  to  the  bottom  of  the  tube  during  centrifuga- 
tion.  The  cotton  plug  may  also  be  replaced  by  a  cap  of  sterile 
tin  foil.  After  careful  balancing,  the  tubes  are  centrifugalized 
at  high  speed  until  the  sediment  is  thrown  down.  The  super- 
natant fluid  is  removed  with  a  sterile  pipette  and  in  this  case 
should  be  preserved  for  the  injection  of  rabbits  for  the  production 
of  precipitins.  The  tubes  are  then  filled  with  sterile  salt  solution, 
the  blood  cells  resuspended  by  drawing  them  in  and  out  of  a 
pipette  and  again  thrown  down  in  the  centrifuge.  This  process 
is  repeated  three  times.  The  washed  sediment  is  then  trans- 
ferred to  another  tube  with  a  graduated  pipette  and  an  equal 
amount  of  salt  solution  added  to  make  the  50  %  suspension  which 

is  used  for  injection. 

» 

2.  PREPARATION  OF  BACTERIAL  EMULSIONS 

Ten  c.c.  of  sterile  salt  solution  are  added  by  means  of  a  pipette 
to  a  24-hour  culture  on  slant  agar  of  the  particular  organism  to 
be  used.  Surface  growth  is  scraped  from  the  agar  by  means  of 
a  platinum  loop  and  the  bacteria  suspended  in  salt  solution  by 
gentle  shaking.  The  suspension  is  then  poured  off  into  a  sterile 
test  tube,  carefully  flaming  the  mouths  both  of  the  culture  tube 
and  the  sterile  test  tube  before  pouring.  The  second  tube  may 
then  be  drawn  out  and  sealed  in  a  blowpipe  flame,  and  the  tube 
entirely  immersed  in  a  water  bath  for  sterilization  ;  or  the  upper 
portion  of  the  tube  may  be  carefully  heated  in  a  Bunsen  flame  to 
kill  any  bacteria  which  may  be  adherent  to  the  side  of  the  upper 
portion,  and  when  the  tube  has  cooled  the  lower  portion  is  then 
immersed  in  a  water  bath,  taking  care  that  the  level  of  the  water 
comes  well  above  the  portion  which  has  not  been  sterilized  by 
flaming.  The  bacteria  should  be  heated  at  60°  C.  for  one  half 
hour  and  are  then  ready  for  injection. 

3.  PREPARATION  OF  SERUM  FOR  INJECTION 

The  serum  or  ascitic  fluid  used  for  the  production  of  precipitins 
should  be  clear  and  sterile  and  is  less  toxic  if  heated  for  30  minutes 
at  56°  C.  before  injection.  It  requires  no  other  preparation. 


6  LABORATORY  COURSE  IN  SERUM  STUDY 

CARE   OF  ANIMALS 

The  animals  used  for  the  routine  work  of  the  course  are  mainly 
guinea  pigs  and  rabbits ;  in  some  cases  dogs  or  cats  are  used,  and 
for  demonstration  a  sheep  is  used  on  one  or  two  occasions. 
Animals  used  should  be  selected  from  among  the  healthy  stock ; 
animals  having  suppurations,  diseased  eyes,  or  skin  diseases 
should  be  discarded.  They  should  be  well  nourished  and  before 
immunization  is  begun  should  be  weighed  and  the  weight  recorded. 
During  the  course  of  treatment  students  should,  if  possible, 
supervise  the  feeding  and  caging  of  their  animals.  Care  should 
be  taken  that  the  cages  are  kept  reasonably  dry,  that  the  animals 
have  plenty  of  water  to  drink  and  the  food  is  abundant,  and 
that  not  too  much  green  feed  is  given.  If  any  of  the  rabbits 
under  treatment  show  a  catarrhal  discharge  from  the  nostrils, 
a  condition  spoken  of  by  animal  dealers  as  "wet  mouth",  these 
animals  should  be  segregated  and  their  nostrils  and  forepaws 
washed  daily  with  weak  bichloride  solution.  Great  care  should 
be  taken  that  the  food  soiled  by  these  animals  is  not  put  into 
cages  of  the  healthy  ones.  Rabbits  and  guinea  pigs  are  best 
preserved  in  a  warm  place,  and  wood  bottoms  on  the  floors  of 
bins  and  cages  are  much  better  than  either  cement  or  tin  unless 
these  are  covered.  They  should  not  be  allowed  to  live  for  days 
on  floors  wet  and  soiled  with  their  own  discharges.  During  the 
immunization  the  animals  should  be  weighed  periodically  and 
their  weight  recorded,  and  if  great  emaciation  and  loss  of  weight 
ensues  in  an  individual  case,  the  animal  should  be  given  a  rest 
and  carefully  fed.  Unless  this  is  done  many  of  the  animals  will 
die  in  the  course  of  immunization. 

There  is  certain  to  be  a  considerable  mortality  among  the 
animals  even  if  the  greatest  care  is  used,  and  it  is  wise  to  start  im- 
munizing two  individuals  for  each  sort  of  substance  used  as  antigen. 

METHODS  OF  INJECTION 
1.     INTRAVENOUS  INJECTION 

When  a  rabbit  is  to  be  injected  the  animal  is  held  by  an  assist- 
ant. The  outer  border  of  the  hairy  side  of  the  ear  is  shaved  and 


8  LABORATORY    COURSE    IN    SERUM    STUDY 

rubbed  vigorously  with  alcohol  until  the  marginal  vein  distends. 
The  needle  of  the  syringe  is  then  introduced  into  the  vein,  point- 
ing toward  the  base  of  the  ear,  and  the  material  injected,  care 
being  taken  to  avoid  the  introduction  of  bubbles.  When  the 
needle  is  withdrawn  a  small  piece  of  absorbent  cotton  is  pressed 
over  the  puncture  to  stop  bleeding.  It  is  well  to  leave  this  cotton 
adherent  to  the  puncture  when  the  animal  is  returned  to  the  cage. 

In  injecting  guinea  pigs  intravenously  it  is  necessary  to  incise 
the  skin  of  the  neck  and  expose  the  external  jugular  vein,  and  unless 
a  very  small  needle  is  used  the  vein  must  be  tied  off  after  injection 
has  been  made.  Etherization  is  needed  for  this  procedure. 

The  injection  of  mice  and  rats  intravenously  is  difficult,  but 
can  be  accomplished  by  rubbing  the  tail  with  xylol  and  holding 
the  mouse  so  that  the  tail  bends  sharply  over  the  edge  of  the  jar. 
The  four  parallel  veins  will  then  stand  out  and  with  care  a  very 
fine  needle  can  be  introduced  into  one  of  them. 

2.  INTRAPERITONEAL  INJECTIONS 

A  rabbit  or  guinea  pig  is  held  back  down  by  an  assistant  and 
a  small  area  of  the  abdominal  wall  median  line  clipped  and  the 
skin  disinfected  with  alcohol  or  iodine.  A  fold  of  skin  and  muscle 
is  pinched  up  with  the  left  hand,  and  the  needle,  which  should  not 
be  too  sharp,  is  cautiously  introduced  directly  into  the  peritoneal 
cavity  and  the  injection  made.  The  safest  point  is  the  median 
line  below  the  umbilicus,  as  in  this  area  there  is  little  danger  of 
puncturing  the  stomach  or  large  intestine. 

3.  SUBCUTANEOUS  INJECTIONS 

Subcutaneous  injections  are  made  in  a  similar  way,  after 
clipping  the  hair  of  the  area  selected  and  pinching  up  a  fold  of 
skin,  taking  care  that  the  needle  does  not  pass  into  the  muscles. 
Rabbits  and  guinea  pigs  are  usually  injected  into  the  surface  of 
the  abdomen,  rats  and  mice  at  the  root  of  the  tail.  If  there  is 
danger  of  the  substance  injected  causing  ulceration,  as  in  the  case 
of  red  cells,  it  is  best  to  inject  rabbits  under  the  skin  of  the  back, 
as  this  area  is  less  likely  to  become  infected. 


10  LABORATORY  COURSE  IN  SERUM  STUDY 

For  injection  a  syringe  is  used  which  will  withstand  boiling. 
For  general  work  all  glass  syringes  of  the  Luer  type  are  the  most 
satisfactory.  A  tightly  fitting  needle  of  gauge  20  to  22  should  be 
used  for "Inrra venous  injections;  for  intraperitoneal  injections 
larger  needles  may  be  used  if  desired.  If  glass  syringes  are  used 
the  needle  may  be  attached  to  the  barrel  of  the  syringe,  the 
plunger  withdrawn,  and  the  two  parts  boiled  separately  for  5 
minutes  in  water  containing  about  1  per  cent  of  sodium  carbonate. 
A  pair  of  forceps  should  be  placed  in  the  sterilizer  with  the  points 
under  the  water.  The  barrel  is  first  removed  with  the  forceps, 
the  plunger  then  removed  and  inserted.  The  fluid  to  be  injected 
is  drawn  into  the  syringe  by  tilting  the  test  tube  or  container 
and  placing  the  beveled  tip  of  the  needle  closely  against  the 
lower  side  of  the  wall.  If  air  is  drawn  into  the  syringe  at  the 
same  time,  it  should  be  expelled  by  holding  the  syringe  vertically, 
needle  up,  covering  the  tip  of  the  needle  with  a  small  piece  of 
cotton  wet  with  alcohol  or  some  other  disinfectant  and  expelling 
the  air.  After  use  the  syringe  should  be  resterilized  if  any 
infected  material  has  been  used,  washed  with  water,  and  the 
needle  washed  with  water,  then  with  alcohol  and  then  with  ether 
to  dry  it  thoroughly.  If  care  is  not  taken  to  clean  the  needles 
thoroughly  after  use,  they  are  certain  to  become  clogged.  If  blood 
or  serum  have  been  injected,  the  needle  and  syringe  must  be 
cleaned  with  cold  water  at  once.  Otherwise  if  alcohol  or  boiling 
water  is  used,  the  protein  will  be  coagulated  and  the  needle  clogged. 
Animals  should  be  labeled  by  attaching  a  number  tag  by  means 
of  a  metal  staple  to  the  ear,  should  be  placed  in  a  cage  with  a  tag 
recording  the  number  of  the  animal,  the  name  of  the  student  in 
charge  and  the  nature  of  the  material  injected,  and  the  date  of 
each  injection. 

SCHEME  FOR  INJECTIONS  (APPROXIMATE  DOSES  AND  INTERVALS) 
1.   Rabbit  immunized  to  B.  typhosus  by  intravenous  injection. 
1st  day  1st  dose  0.05  agar  slant 
6th  day  2d   dose  0.1    agar  slant  heated  as 

llth  day  3d   dose  0.2    agar  slant  indicated 

16th  day  4th  dose  0.2    agar  slant  below 

24th  day  5th  dose  0.2    agar  slant 


12  LABORATORY  COURSE  IN  SERUM  STUDY 

2.     Rabbit  immunized  to  B.  typhosus  by  subcutaneous  injections. 
Injections  at  five  or  six  day  intervals,  followed  by  intravenous  injections. 


1st  dose  0.1  agar  slant 
2d  dose  0.2  agar  slant 
3d  dose  0.3  agar  slant 
4th  dose  0.5  agar  slant 
5th  dose  0.5  agar  slant 


3.  Rabbit  immunized  to  B.  typhosus  and  to  B.  coli  communior  by 
intravenous  injection.     Intervals  as  above. 

1st  dose  0.05  agar  slant  of  B.  typhosus 
2d  dose  0.1  agar  slant  of  B.  typhosus 
3d  dose  0.2  agar  slant  of  B.  typhosus 
4th  dose  0.2  agar  slant  of  B.  typhosus,  plus  1 

0.05  agar  slant  of  B.  coli  communior  J 
5th  dose  0.2    agar  slant  of  B.  typhosus,  plus 

0.05  agar  slant  of  B.  coli  communior 
6th  dose  0.2    agar  slant  of  B.  typhosus,  plus  j 

0.1    agar  slant  of  B.  coli  communior  J 
7th  dose  0.2    agar  slant  of  B.  typhosus,  plus  1 

0.2    agar  slant  of  B.  coli  communior  J 

4.  Guinea  pig  immunized  to  Sp.  cholerse  by  intraperitoneal  injec- 
tion.    An  old  laboratory  strain  of  the  cholera  vibrio  should  be  used 
for  immunization.     Intervals  as  above. 

1st  dose  0.05  agar  slant 
2d  dose  0.1  agar  slant 
3d  dose  0.2  agar  slant 
4th  dose  0.2  agar  slant 
5th  dose  0.2  agar  slant 

5.  Rabbit  immunized  to  micrococcus  aureus  by  intravenous  injec- 
tion.    Intervals  as  above.     In   the   case  of  this  microorganism   the 
intervals  should  be  lengthened  if  there  is  any  indication  of  illness. 
Great  care  is  necessary  in  avoiding  overdosage. 

1st  dose  0.05  agar  slant 
2d  dose  0.1  agar  slant 
3d  dose  0.2  agar  slant 
4th  dose  0.2  agar  slant 
5th  dose  0.2  agar  slant 


14          LABORATORY  COURSE  IN  SERUM  STUDY 

6.  Rabbit  immunized  to  sheep  serum  by  intravenous  injection. 
Five-day  intervals. 

1st  dose  2.0  c.c. 
2d  dose  2.0  c.c. 
3d  dose  2.0  c.c. 

7.  Rabbit  immunized  to  human  serum  (ascitic  fluid)  by  intravenous 
injection.     Five-day  intervals. 

1st  dose  4.0  c.c. 
2d  dose  4.0  c.c. 
3d  dose  4.0  c.c. 

8.  Rabbit  immunized  to  horse  serum.     (Immunize  noteless  than 
three  in  order  to  obtain  sufficient  serum  for  later  exercises.) 

1st  dose  2.0  c.c. 
2d  dose  2.0  c.c. 
3d  dose  2.0  c.c. 

9.  Rabbit  immunized  to  sheep  erythrocytes  by  intravenous  injec- 
tion.    Four  or  five  day  intervals. 

1st  dose  4.0  c.c.  of  50  %  suspension 
2d  dose  4.0  c.c.  of  50  %  suspension 
3d  dose  4.0  c.c.  of  50  %  suspension 

A  fourth  injection  may  be  given  in  some  cases,  but  is  not  often 
necessary. 

10.  Rabbit  immunized  to  sheep  erythrocytes  by  intraperitoneal 
injections.     Intervals  as  above. 

1st  dose  4.0  c.c.  of  50  %  suspension 
2d  dose  8.0  c.c.  of  50  %  suspension 
3d  dose  8.0  c.c.  of  50  %  suspension 
4th  dose  8.0  c.c.  of  50  %  suspension 

11.  Rabbit  immunized  to  cat  erythrocytes  by  intravenous  injec- 
tions of  2  c.c.  at  4-day  intervals.  —  See  Lesson  XII. 

A  preliminary  titration  of  the  serum  done  with  a  specimen 
obtained  from  an  ear  vein  should  be  done  about  the  eighth  or 
ninth  day  after  the  last  injection.  If  this  is  satisfactory,  the 
bleeding  of  the  animal  is  usually  done  the  ninth  or  tenth  day.  No 
absolute  rule  can  be  established  as  to  exact  period  at  which  the 
serum  has  the  highest  antibody  concentration,  this  depending  to 


16  LABORATORY  COURSE  IN  SERUM  STUDY 

some  extent  on  the  health  of  the  animal,  the  size  of  the  injec- 
tions, and  the  intervals.  It  is  a  safe  rule  to  assume  that  the 
high  point  in  the  curve  is  apt  to  lie  between  the  eighth  and  the 
twelfth  day  after  the  last  injection  and  to  be  guided  by  this  in 
preliminary  titrations. 

THE  BLEEDING  OF  ANIMALS 

Before  the  immunization  of  the  animal  is  begun  a  sample  of 
blood  should  be  taken  from  the  ear  vein  of  the  animal  and  the 
serum  stored  in  the  refrigerator  for  use  as  a  control  at  a  later  exer- 
cise when  the  serum  of  this  animal  is  tested  for  immune  bodies. 
The  blood  can  be  obtained  from  the  marginal  vein  in  the  ear  in 
the  following  manner. 

The  animal  is  firmly  held  with  the  head  down  by  an  assistant. 
The  lateral  portion  of  the  hairy  surface  of  the  ear  is  carefully 
shaved  and  the  skin  rubbed  vigorously  to  cause  the  veins  to  dis- 
tend. If  necessary  the  ear  may  be  rubbed  with  xylol.  The  skin 
over  the  vein  is  washed  over  with  alcohol  and  a  cut  made  in  the 
vein  with  a  razor  blade  or  other  sharp  instrument.  The  blood  is 
allowed  to  drip  into  a  clean  centrifuge  tube  until  about  4  c.c. 
(60  drops)  are  obtained.  It  is  then  allowed  to  coagulate  and  the 
edges  of  the  clot  are  freed  from  the  side  of  the  tube  by  means  of  a 
sterile  platinum  wire.  The  tube  is  centrifugalized  and  the  clear 
serum  thus  expressed  is  transferred  to  a  sterile  test  tube  by 
means  of  a  capillary  pipette  with  a  rubber  nipple  attached.  It  is 
then  labelled  carefully  and  stored  in  the  icebox  for  future  use. 

Later  bleeding  from  the  ear  veins  of  a  rabbit  can  be  facilitated 
by  strapping  the  animal  down  on  a  flat,  hot  water  bag.  A  techni- 
cal point  of  great  simplicity  but  which  helps  to  make  the  sort  of 
cut  in  the  vein  which  favors  free  bleeding,  consists  in  taking  a 
pointed  steel  writing  pen,  breaking  off  one  of  the  prongs  and 
plunging  the  stilette  thus  made  straight  into  the  vein  in  a  direc- 
tion toward  the  heart.  The  entry  thus  made  through  the  wall 
of  the  vein  does  not  easily  collapse  and  very  free  bleeding  results. 

To  bleed  a  rabbit  from  the  carotid  the  rabbit  is  strapped  down 
on  its  back  on  a  dissecting  board  and  ether  administered.  A 


18  LABORATORY  COURSE  IN  SERUM  STUDY 

central  incision  along  the  neck  above  the  trachea  is  made,  and  the 
carotid  will  be  found  lying  very  close  to  the  trachea  on  either  side. 
A  ligature  is  applied  distally,  the  artery  raised  on  the  handle  of  a 
forceps  and  a  bulldog  clamp  applied  toward  the  ridge  of  the  neck. 
A  sterile  glass  canula  can  be  inserted,  but  we  have  found  that 
with  a  little  skill  it  is  quite  easy  to  lift  up  the  artery  and  hold  it 
by  its  wall  with  a  forceps  in  such  a  way  that  the  blood  stream 
is  not  blocked.  The  vessel  is  then  cut,  the  clamp  removed, 
and  by  aiming  the  artery  with  the  forceps  in  which  it  is  held  the 
stream  of  blood  can  be  directed  into  a  sterile  test  tube.  In  this 
way  rabbits  are  usually  bled  until  distinct  respiratory  distress 
ensues  and  the  stream  of  blood  ceases,  the  blood  coming  in 
drops.  The  clamp  is  then  applied,  the  vessel  tied  and  the  rabbit 
sewed  up  aseptically.  Such  a  rabbit  can  be  kept  alive  and 
further  used  after  recovery  for  continuation  of  the  immunization.1 

The  bleeding  of  guinea  pigs  from  the  carotid  is  in  every  way 
similar,  though  a  little  more  difficult  than  the  bleeding  of  rabbits. 
When  guinea  pigs  are  bled,  as  they  will  be  for  the  purpose  of  ob- 
taining complement  or  alexin,  it  is  necessary  to  do  this  carefully 
in  order  to  obtain  the  best  results.  The  blood  is  best  taken  into 
large  sterile  test  tubes  with  as  little  violence  as  possible  in  order 
that  there  may  be  no  breaking  up  of  blood  cells  with  consequent 
hemolysis.  It  is  best  to  take  the  blood  the  evening  before  it  is 
to  be  used,  to  allow  it  to  clot  in  the  test  tube  and  then  to  separate 
the  clot  very  gently  from  the  sides  of  the  tube.  The  tube  should 
then  be  set  aside  in  the  refrigerator  at  a  low  temperature  (1-4°  C.). 
The  rapidity  with  which  complement  degenerates  at  higher  tem- 
peratures is  generally  underestimated.  The  following  day  the 
serum  is  pipetted  away  from  the  clot. 

Some  observers  believe  that  complement  increases  in  potency 
on  standing  for  some  hours  on  the  clot.  This  was  the  con- 
tention of  Henderson-Smith,  though  it  has  recently  been  con- 
tradicted by  Addis  and  others.  It  is  a  good  practice  to  obtain  it 
in  this  way,  however,  because  blood  so  taken  is  apt  to  be  clearer 
and  freer  from  haemolysis  than  blood  centrifugalized  immediately 
after  clotting.  Although  a  slight  tinge  of  haemolysis  does  not 

1  Intravenous  injections  of  10  to  20  c.c.  of  warm  salt  solution  will  often  help  to 
keep  such  animals  alive. 


\ 


20  LABORATORY   COURSE   IN   SERUM   STUDY 


f-'-1-  v/ 

^bes 

^       •  u~ 


render  blood  useless  for  haemolytic  purposes,  nevertheless  the 
products  of  haemolysis  do  affect  the  haemolytic  reaction  and  it  is 

t  to  have  an  entirely  clear  alexin  for  use. 

The  technique  of  bleeding  of  sheep  or  other  large  animals  has 
been  described  on  page  2. 

LESSON   II 
BACTERICIDAL   POWER    OF   NORMAL    SERUM 

NORMAL  blood  plasma  or  serum  possesses  the  power  of  killing 
bacteria.  Not  all  bacteria  are  equally  susceptible  to  this  effect. 
Some,  like  the  Gram-positive  cocci,  the  anthrax  bacillus,  and 
others,  are  probably  killed  in  the  circulation  only  by  the  co- 
operation of  serum  and  leucocytes  in  phagocytosis.  When  the 
serum  is  heated  to  56°  C.,  its  bactericidal  power  is  suspended. 

Reagents : 

1.  Fresh  normal  rabbit  serum,  unheated. 

2.  Bacillus  typhosus  —  24-hour  broth  culture    diluted    1-5000  in 
sterile  salt  solution. 

3.  Sterile  salt  solution. 

The  materials  in  this  experiment  must  be  measured  with  sterile 
pipettes  and  placed  in  sterile  test  tubes,  with  care  to  avoid  con- 
tamination during  the  process  of  the  experiment. 

(a)  Place  0.5  c.c.  of  normal  rabbit  serum  in  each  of  two  sterile 
test  tubes  and  heat  one  tube  for  half  an  hour  at  56°  C.  Then 
set  up  the  following  preparations  in  sterile  plugged  test  tubes : 

1.  Normal  rabbit  serum  0.5  c.c.  +  Typhoid  broth  (1-5000)  0.5  c.c.1 

(fresh) 

2.  Normal  rabbit  serum  0.5  c.c.  +  Typhoid  broth  (1-5000)  0.5  c.c. 

(heated) 

3.  Salt  solution  0.5  c.c.  +  Typhoid  broth  (1-5000)  0.5  c.c. 

1  To  make  dilutions  for  this  and  other  purposes  proceed  as  follows : 

1.  1  c.c.  of  original  substance  to  9  c.c.  of  salt  solution  =  1-10 

2.  1  c.c.  of  dilution  (1.)  to  9  c.c.  of  salt  solution  =  1-100 

3.  1  c.c.  of  dilution  (2.)  to  4  c.c.  of  salt  solution  =  1-500 

4.  1  c.c.  of  dilution  (3.)  to  1  c.c.  of  salt  solution  =  1-1000 

5.  1  c.c.  of  dilution  (4.)  to  4  c.c.  of  salt  solution  =  1-5000 

This  illustrates  the  general  method,  which  can  be  shortened  or  lengthened 
by  longer  or  smaller  intervals  as  the  particular  occasion  requires. 


22  LABORATORY  COURSE  IN  SERUM  STUDY 

(6)  Place  the  tubes  in  the  incubator  for  two  hours,  and  then 
pour  into  each  tube  a  tube  of  agar  which  has  been  melted  in 
boiling  water  and  cooled  to  42°  C.  Pour  the  entire  contents 
of  each  tube  into  a  sterile  Petri  dish.  When  the  plates  are 
hardened,  place  them  in  the  incubator  in  an  inverted  position, 
to  be  observed  at  the  following  lesson.  The  plates  made  from 
tubes  2  and  3  will  show  an  enormous  number  of  minute 
typhoid  colonies  after  incubation.  The  plate  from  tube  1  will 
be  sterile  or  may  show  a  few  large  colonies. 

ILEMOLYTIC    AND    ILEMAGGLUTINATIVE    POWER    OF 
NORMAL   SERUM 

The  normal  blood  serum  of  many  animals  has  haemolytic 
and  haemagglutinative  action  upon  the  red  blood  cells  of  animals 
of  some  other  species.  This  hsemolytic  action,  like  the  bacteri- 
cidal action,  is  lost  when  the  serum  is  heated  to  56°  C.  The 
haemagglutinating  property,  on  the  other  hand,  is  relatively 
resistant  to  heat  and  is  not  destroyed  until  the  serum  is  heated  to 
70°  C.  or  above.  Such  a  hsemolytic  normal  serum  acts  only  on 
the  erythrocytes  of  certain  other  species,  sometimes  upon  2  or  3 
varieties  with  differing  intensity.  Thus  goat  serum  is  strongly 
haemolytic  for  rabbit  cells  —  less  so  for  those  of  guinea  pigs. 
Dog  serum  contains  a  relatively  large  amount  of  normal  haemo- 
lysin  for  most  cells  used  in  laboratory  work.  Guinea  pig  cells 
are  highly  susceptible,  and  human  cells  relatively  resistant  to 
haemolysis  by  dog  serum. 

Reagents : 

1.  Fresh  dog  serum. 

2.  2  %  suspension  of  washed  guinea  pig  cells. 

3.  2  %  suspension  of  washed  human  cells. 

4.  Salt  solution. 

Place  0.25  c.c.  of  dog  serum  in  a  test  tube  and  heat  for  one  half 
hour  in  water  bath  at  56°  C.  Bring  up  volume  to  2.5  c.c.  with  normal 
salt  solution.  Prepare  a  similar  1-10  dilution  of  unheated  dog  serum. 
Set  up  the  following  experiment  in  half-inch  tubes : 


24          LABOKATORY  COURSE  IN  SERUM  STUDY 

1.  Dog  serum  unheated  (1-10)  0.5  c.c.  +  Guinea  pig  cells  2  %  0.5  c.c. 

2.  Dog  serum  unheated  (1-10)  0.5  c.c.  +  Human  cells  2  %  0.5  c.c. 

3.  Dog  serum  heated  (1-10)      0.5  c.c.  +  Guinea  pig  cells  2  %  0.5  c.c. 

4.  Dog  serum  heated  (1-10)      0.5  c.c.  +  Human  cells  2  %  0.5  c.c. 

5.  Salt  solution  0.5  c.c.  +  Guinea  pig  cells  2  %  0.5  c.c. 

6.  Salt  solution  0.5  c.c.  -f  Human  cells  2  %  0.5  c.c. 

Incubate  the  tubes  one  hour  at  37°  C.  in  water  bath,  observing 
changes  in  cells  at  intervals  of  5,  10,  15,  30,  and  60  minutes.  Tabulate 
the  results.  Tube  1  should  show  prompt  haemolysis;  tube  3  should 
show  agglutination  of  the  cells.  In  tubes  2  and  4  there  should  be  no 
observable  change  in  the  cells  in  most  samples  of  dog  serum. 

Each  student  needs:  10  J^-inch  test  tubes,  test  tube  rack,  5  one-c.c. 
pipettes,  1  five-c.c.  pipette. 

LESSON  III 

HAEMOLYSIS.     (EHRLICH    AND    MORGENROTH    EXPERI- 
MENTS) 

1.  To  a  group  of  students  is  assigned  the  task  of  bleeding  a  sheep 
from  the  jugular  vein  into  a  flask  containing  glass  beads.     After  defibrina- 
tion  the  cells  are  washed  three  times  in  salt  solution  and  a  5  per  cent 
suspension  of  the  well-packed  sediment  is  made  (1.0  c.c.  of  sediment  and 
19.0  c.c.  of  salt  solution). 

2.  To  another  group  of  students  is  assigned  the  bleeding  of  guinea 
pigs  from  the  carotid  artery  into  centrifuge  tubes.     The  blood  is  allowed 
to  clot,  this  clot  is  "rimmed"  and  the  serum  separated.     The  process 
may  be  hurried  by  centrifugation.     The  serum  is  then  diluted,  1  part  of 
serum  to  9  parts  of  salt  solution. 

3.  Immune  rabbit  serum  (lytic  for  sheep  cells)  will  be  given  out 
diluted.1    This  serum  has  been  heated  for  one  half  hour  at  56°  C. 
before  dilution,  in  order  to  destroy  its  alexin  or  complement.     This  is 
done  so  that  no  unknown  amount  of  complement  may  be  present  in  the 
final  tests. 

All  these  materials  must  be  prepared  before  the  regular  class  period, 
as  the  experiments  themselves  consume  several  hours.  Provided  the 
guinea  pig  serum  is  kept  very  cold  (0°-^i0  C.),  all  the  materials  can  be 
prepared  the  preceding  day. 

1  In  order  to  save  time  in  the  course  an  immune  serum  previously  prepared  by 
the  instructor  is  used  here.  The  immune  haemolytic  serum  whose  preparation 
was  begun  by  the  students  in  Lesson  I  is  to  be  titrated  later  and  used  for  the 
lessons  on  complement  fixation . 


26          LABORATORY  COURSE  IN  SERUM  STUDY 

Experiment  1 

To  SHOW  THAT  RED  BLOOD  CELLS  WILL  ABSORB  AMBOCEPTOR  BUT 

NOT   COMPLEMENT 

Set  up  two  mixtures  in  centrifuge  tubes  as  indicated  below: 

A.  Sheep  cells  and  heated  immune  rabbit  serum 

B.  Sheep  cells  and  fresh  normal  guinea  pig  serum. 

Incubate  for  30  minutes  at  37  C.,  then  centrifugalize  and  remove 
the  supernatant  fluid,  using  a  capillary  pipette  with  rubber  nipple. 

Wash  the  sediment  once  in  salt  solution. 

Resuspend  the  washed  sediment  in  each  tube  in  3  c.c.  salt  solution. 

Add  the  reagents  as  indicated  and  incubate  for  one  hour  at  37  C., 
observing  at  short  intervals.  Tabulate  the  results. 

Tube  A 

Sheep  cells  5% 1.0  c.c. 

Inactivated  immune  rabbit  serum l      ....     1.0  c.c. 

Incubate  30  minutes  at  37°  C.,  centrifugalize,  remove  the  superna- 
tant fluid  to  another  tube  and  wash  the  sediment. 

We  now  have  2  tubes  —  one  (Tube  1)  containing  the  supernatant 
fluid,  the  other  (Tube  2)  the  sediment  from  the  original  Tube  A.  To 
these  tubes  add  reagents  as  follows : 

* 

Tube  1  Tube  2 

(Supernatant  fluid  from  (Sediment   from  Tube   A 

Tube  A) 2.0  c.c.  resuspended      in      salt 

Guinea  pig  serum  (1-10)  1.0  c.c.        solution) 3.0  c.c. 

Sheep  cells  5%     .     .     .  1.0  c.c.  Guinea  pig  serum  (1-10)     1.0  c.c. 

Incubate  and  observe. 

In  which  of  the  two  does  haemolysis  take  place?  What  conclu- 
sion as  to  the  reaction  that  has  taken  place  in  the  original  mixture  in 
Tube  A  can  you  draw  from  this? 

TubeB 

Sheep  cells  5% 1.0  c.c. 

Guinea  pig  serum  (1-10) 1.0  c.c. 

Incubate  30  minutes  at  37°  C.,  centrifugalize,  remove  supernatant 
fluid  to  another  tube  and  wash  sediment. 

1  Diluted  by  the  instructor  so  as  to  contain  two  hemolytic  units  in  1  c.o. 


28          LABORATORY  COURSE  IN  SERUM  STUDY 

Then,  as  in  the  preceding  case,  we  have  two  tubes  to  which  the 
following  additions  are  made  : 

Tube  1  Tube  2 

(Supernatant  fluid  from  (Sediment  from    Tube  B 

Tube  B)  about  .     .     .  2.0  c.c.  resuspended      in      salt 

Immune  rabbit  serum  1.0  c.c.        solution) 3.0  c.c. 

Sheep  cells  5%       .     .  1.0  c.c.  Immune  rabbit  serum  .     1.0  c.c. 

Incubate  and  observe. 

In  which  tube  does  haemolysis  take  place?     Contrast  this  with 
results  in  Tube  A  and  draw  conclusions. 


Experiment  2 

TO  SHOW  THAT  AT  0°  C.  CELLS  WILL  ABSORB  AMBOCEPTOR  ALONE 
FROM  A  MIXTURE  OF  AMBOCEPTOR  AND  COMPLEMENT 

The  same  reagents  will  be  used  in  this  as  in  the  preceding  experi- 
ment. The  three  reagents  will  first  be  cooled  in  ice  water  and  then  be 
mixed  in  a  cold  centrifuge  tube,  and  allowed  to  stand  at  0°  C.  for  an 
hour.  With  proper  care  this  can  easily  be  done  in  a  battery  jar  filled 
with  cracked  ice  and  brine. 

The  success  of  the  experiment  depends  on  having  the  reagents  and 
the  centrifuge  tube  and  metal  holder  thoroughly  cooled  in  ice  water 
before  the  mixture  is  made  and  in  centrifugalizing  and  removing  the 
supernatant  fluid  before  they  have  time  to  regain  the  temperature  of 
the  room.  The  protocol  for  this  experiment  is  given  below : 

Place  centrifuge  tube  in  holder  in  a  cup  of  cracked  ice  and  water  and 
add: 

5%  sheep  cells 1.0  c.c. 

Inactive  immune  rabbit  serum  (diluted)  .     .     1.0  c.c. 
Guinea  pig  serum  (1-10) 1.0  c.c. 

After  twenty  minutes  the  tube  is  centrifugalized,  the  supernatant 
fluid  removed  and  half  of  it  placed  in  each  of  two  small  test  tubes. 
The  sediment  is  to  be  washed  once  in  cold  salt  solution,  resuspended 
in  4  c.c.  of  salt  solution  and  half  of  this  suspension  placed  in  each  of 
two  small  test  tubes. 

We  now  have  four  tubes  —  two  of  which  contain  washed  sediment 
and  two  of  which  contain  supernatant  fluid  of  the  original  mixture. 
To  these  the  following  additions  are  now  made : 


30          LABORATORY  COURSE  IN  SERUM  STUDY 

Tube  1 

Supernatant  fluid 1.5  c.c. 

Fresh  guinea  pig  serum  1-10  (complement)  0.5  c.c. 

Washed  sheep  cells  5  % 0.5  c.c. 

Tube  2 

Supernatant  fluid 1.5  c.c. 

Immune  rabbit  serum        0.5  c.c. 

Washed  sheep  cells  5  % 0.5  c.c. 

TubeS 

Sediment 2.0  c.c. 

Guinea  pig  serum  1-10 0.5  c.c. 

Tube  4 

Sediment 2.0  c.c. 

Immune  rabbit  serum 0.5  c.c. 

Incubate  one  hour  at  37°  C.,  observing  at  frequent  intervals. 

The  results  will  show  that  the  haemolytic  amboceptor  or  sensitizer 
present  in  the  original  heated  immune  serum  was  bound  by  the  cells  at 
0°  C.  —  but  at  this  low  temperature  the  complement  or  alexin  remained 
not  only  inactive  but  unbound  and  free. 

APPARATUS 
Each  student  will  require : 

3  graduated  centrifuge  tubes 

8  half-inch  test  tubes 

9  one  c.c.  pipettes 
1  test  tube  rack 

1  bottle  of  salt  solution 

1  jar  of  cracked  ice  (pint  measure) 

1  rubber  nipple  and  capillary  pipette 

The  class  will  require  : 
1  outfit  for  bleeding  sheep : 
needle, 
tourniquet, 
razor  and  scissors, 
bottle  of  tincture  of  iodine, 
sterile  flask  containing  beads. 


32          LABORATORY  COURSE  IN  SERUM  STUDY 

1  or  more  outfits  for  bleeding  guinea  pigs : 

scissors, 
forceps, 
ether  cone, 
centrifuge  tubes. 

MATERIALS 
Each  student  will  require : 

Washed  sheep  cells  5  % 6.0  c.c. 

Guinea  pig  serum  1-10 6.0  c.c. 

Inactive  serum  of  rabbit  immunized  against 
sheep  cells,  diluted  so  that  each  c.c.  con- 
tains 2-4  hsemolytic  doses 6.0  c.c. 


LESSON  IV 

QUANTITATIVE  RELATIONSHIPS  OF  AMBOCEPTOR  AND 

COMPLEMENT 

METHODS  OF  TITRATION 

THE  haemolysis  of  red  cells  by  specific  sensitizer  [or  ambocep- 
tor]  and  alexin  [or  complement]  is  a  delicately  quantitative 
reaction  in  which  very  definite  amounts  of  the  three  substances 
are  necessary  to  produce  a  complete  reaction.  As  a  unit  of  red 
cells,  one  cubic  centimeter  of  a  5  per  cent  suspension  of  red 
cells  is  conveniently  taken.  This  means  that  the  cells  after 
washing  are  sedimented  in  the  centrifuge  and  1.0  c.c.  of  the 
sediment  is  added  to  19.0  c.c.  of  salt  solution.  (Some  investi- 
gators have  worked  with  suspensions  representing  5  per  cent  of 
the  original  blood.  Assuming  the  cells  to  constitute  50  per 
cent  of  the  blood  volume,  suspensions  so  made  up  are  just  half 
as  concentrated  as  the  ones  here  used.  In  all  the  work  here  de- 
scribed the  term  5  per  cent  suspension  is  taken  to  represent  5  per 
cent  by  actual  volume  of  erythrocyte  sediment  in  salt  solution.) 

Definite  amounts  of  sensitizer  and  of  alexin  are  necessary  to 
lake  completely  one  cubic  centimeter  of  such  a  suspension,  and  it 
is  necessary  for  accurate  experimentation  to  determine  for  each 
set  of  reagents  the  minimal  amounts  necessary  to  accomplish 


34  LABORATORY   COURSE    IN   SERUM   STUDY 

this  purpose.  It  must  be  remembered  that  no  two  suspensions 
of  red  cells  are  exactly  alike  —  since  it  is  impossible  to  measure 
them  with  absolute  accuracy,  and  since  the  resistance  of  erythro- 
cytes  to  haemolysis,  even  when  taken  from  the  same  animal  on 
different  days,  may  vary.  No  two  fresh  sera,  moreover,  are 
entirely  alike  in  alexic  activity.  The  experiments  given  below 
will  demonstrate  the  method  of  determining  by  titration  the 
minimal  haemolytic  dose  of  complement  and  amboceptor ;  they 
will  also  demonstrate  that  this  dose  varies  reciprocally,  that  is, 
that  with  a  small  amount  of  complement  a  relatively  larger 
amount  of  amboceptor  will  be  necessary  to  cause  complete 
haemolysis,  whereas  with  a  large  amount  of  complement  only  a 
small  amount  of  amboceptor  is  necessary.  There  is,  however,  no 
simple  proportionality. 

Experiment  1 

TITRATION  OF  H^MOLYTIC  SENSITIZER  OR  AMBOCEPTOR.     DETERMINA- 
TION OF  UNIT 

The  unit  of  sensitizer,  or  amboceptor,  is  the  smallest  amount 
which  will  cause  complete  laking  of  a  unit  of  cells  (1.0  c.c.  of  a 
5  per  cent  emulsion)  1  in  one  hour,  in  the  presence  of  an  excess  of 
complement. 

As  the  degree  of  dilution  is  an  important  factor  it  is  necessary  that 
the  volume  of  all  the  tubes  be  brought  to  a  uniform  standard  before 
the  reaction  actually  begins.  For  this  reason  the  reagents  are  added  in 
the  following  order : 

1.  Sensitizer  [amboceptor] 

2.  Alexin  [complement] 

3.  Saline  to  make  all  volumes  equal 

4.  Cells 

A  control  tube  must  be  set  up  to  prove  that  the  amount  of  alexin  or 
complement  used  will  not  of  itself  lake  the  cells. 

1  For  convenience  or  economy  0.5,  0.25  or  0.1  c.c.  of  5  per  cent  red  cells  are 
sometimes  taken  as  the  unit,  and  in  such  cases  the  unit  of  amboceptor  and  com- 
plement must  be  proportionately  reduced.  For  the  sake  of  uniformity  results 
so  obtained  can  easily  be  transposed  to  terms  of  the  1  c.c.  unit,  as  is  often  done 
in  reporting  Wassermann  tests. 


C6 


LABORATORY   COURSE    IN   SERUM   STUDY 


Add  saline 
to  2  c.c. 
and  then 
0.5  c.c.  of 
the  red  cell 
emulsion. 1 


Set  up  the  following  tests : 

Series  1 

Immune  Serum  (Sensitizer)  Complement 

Tube  1.     (diluted  1-100)    0.5  c.c.  0.05  c.c. 

2.  (diluted  1-100)    0.2  c.c.  0.05  c.c. 

3.  (diluted  1-1000)  1.0  c.c.  0.05  c.c. 

4.  (diluted  1-1000)  0.5  c.c.  0.05  c.c. 

5.  (diluted  1-1000)  0.4  c.c.  0.05  c.c. 

6.  (diluted  1-1000)  0.3  c.c.  0.05  c.c. 

7.  (diluted  1-1000)  0.2  c.c.  0.05  c.c. 

8.  (diluted  1-1000)  0.1  c.c.  0.05  c.c. 

9.  (diluted  1-1000)  0.0  c.c.  0.05  c.c. 

(Complement  Control) 

Series  2 

Immune  Serum  (Sensitizer)  Complement 

Tube  1.     (diluted  1-100)    0.5  c.c.  0.02  c.c. 

2.  (diluted  1-100)    0.2  c.c.  0.02  c.c. 

3.  (diluted  1-1000)  1.0  c.c.  0.02  c.c.     Add  saline 

4.  (diluted  1-1000)  0.5  c.c.  0.02  c.c.     to  2  c.c. 

5.  (diluted  1-1000)  0.4  c.c.  0.02  c.c.     and  then 

6.  (diluted  1-1000)  0.3  c.c.  0.02  c.c.     0.5  c.c.  of 

7.  (diluted  1-1000)  0.2  c.c.  0.02  c.c.     the  red  cell 

8.  (diluted  1-1000)  0.1  c.c.  0.02  c.c.     emulsion. 

9.  (diluted  1-100)    0.5  c.c.  0.00  c.c.  ( 

(Sensitizer  Control) 

Series  3 

Immune  Serum  (Sensitizer)  Complement 

Tubel.     (diluted    -100)    0.5  c.c.  0.01  c.c. 

2.  (diluted    -100)    0.2  c.c.  0.01  c.c. 

3.  (diluted    -1000)  1.0  c.c.  0.01  c.c.     Add  saline 

4.  (diluted    -1000)  0.5  c.c.  0.01  c.c.     to  2  c.c. 

5.  (diluted    -1000)  0.4  c.c.  0.01  c.c.     and  then 

6.  (diluted    -1000)  0.3  c.c.  0.01  c.c.     0.5  c.c.  of 

7.  (diluted  1-1000)  0.2  c.c.  0.01  c.c.     the  red  cell 

8.  (diluted  1-1000)  0.1  c.c.  0.01  c.c.     emulsion. 

9.  (diluted  1-1000)  0.0  c.c.  0.00  c.c.  J 

(Salt  Solution  Control) 

1  A  half  c.c.  instead  of  one  c.c.  is  used  in  order  to  save  materials.  The  amounts 
of  complement  and  amboceptor  needed  are  of  course  half  those  that  would  be  needed 
for  1  c.c.  of  cells. 


38 


LABORATORY   COURSE    IN   SERUM   STUDY 


In  measuring  0.05,  0.02  and  0.01  c.c.  of  complement  it  is  best  to  use 
a  half  c.c.,  .2  c.c.  and  .1  c.c.  of  a  1-10  dilution.  Shake  each  tube 
thoroughly  when  all  the  reagents  have  been  added.  Incubate  one  hour 
in  water  bath  at  37°  C.  and  record  by  -f-  or  —  signs  haemolysis  at  15, 
30  and  60  minutes  in  the  following  protocol: 


IMMUNE  SERUM  (SENSITIZER  OR  AMBOCEPTOR) 

Diluted  1-100 

Diluted  1-1000- 

0.5 

0.2 

1.0 

0.5 

0.4 

0.3 

0.2 

0.1 

0.0 

Complement  0.05    . 
Complement  0.1 
Complement  0.2 

Indicate  what  the  unit  is  in  this  case. 

The  tabulation  given  for  final  record  is  the  manner  in  which  an 
experiment  of  this  kind  is  best  recorded  by  experienced  workers.  We 
have  inserted  the  simpler  protocols  of  the  three  series  before  this  in 
order  to  make  the  purpose  of  this  experiment  and  its  execution  a  little 
more  easy. 

(On  account  of  the  costliness  of  guinea-pig  serum  students  should 
divide  themselves  into  groups  of  3,  of  whom  each  student  should  set 
up  one  series  of  amboceptor  dilutions  with  one  of  the  complement  quan- 
tities.) 

Individual  guinea  pigs  vary  in  the  complementary  activity  of 
their  serum.  For  accurate  hsemolytic  experiments,  however,  it 
is  necessary  to  use  a  constant  amount  of  complement  activity. 
For  this  reason  the  unit  of  amboceptor  (which  if  carefully  pre- 
served remains  constant  over  long  periods)  is  taken  as  a  standard 
to  which  the  strength  of  each  fresh  complement  serum  is  adjusted. 

Strictly  speaking,  the  unit  of  complement  should  be  that 
amount  which  gives  complete  laking  with  one  unit  of  cells  and 
one  unit  of  amboceptor.  In  practice,  however,  this  turns  out  to 
be  too  large  an  amount,  and  for  this  reason  complement  is  usually 
titrated  with  two  units  of  amboceptor.  The  unit  of  complement 
then  may  be  defined  as  the  least  amount  that  completely  hemo- 
lyses  1.0  c.c.  of  a  five  per  cent  emulsion  of  red  cells  in  the  presence 
of  two  units  of  amboceptor  in  thirty  minutes  in  the  water  bath. 
If  the  test  is  done  with  smaller  quantities  of  red  cells  the  unit  is 
correspondingly  smaller.  Set  up  titrations  with  both  two  and 
one  units  as  follows: 


40 


LABORATORY  COURSE  IN  SERUM  STUDY 


TUBE 

COMPLEMENT  1-10 

AMBOCEPTOR 

1 

0.5  c.c. 

2  units   ' 

2 

0.4 

2  units 

Add 

3 

0.3 

2  units 

Saline  to  2  c.c. 

4 

0.2 

2  units 

5%  cells,  0.5  c.c. 

5 

0.1 

2  units  t 

6 

0.5 

1  unit 

7 

0.4 

1  unit 

Add 

8 

0.3 

1  unit 

Saline  to  2  c.c. 

9 

0.2 

1  unit 

5%  cells,  0.5  c.c. 

10 

0.1 

1              1  unit 

Incubate.    Read  results  at  15,  30  and  60  minutes. 

(Indicate  unit  found.) 

It  should  be  remembered  that  even  with  these  two  reagents,  com- 
plement and  amboceptor,  titrated,  the  amounts  determined  might  vary 
somewhat  if  cells  from  another  sheep  or  taken  from  this  sheep  on  another 
day  were  used  —  since  the  resistance  of  the  cells  is  also  a  variable 
factor. 

In  many  experiments  it  is  expedient  to  use  red  cells  previously 
sensitized  with  a  definite  number  of  amboceptor  units.  In  order  to  do 
this  the  following  method  is  employed.  The  red  cells  after  washing  are 
taken  up  in  salt  solution  in  a  definite  concentration  which  for  ordinary 
work  is  5  per  cent.  Inactivated  immune  serum  (amboceptor  or  sensi- 
tizer)  diluted  in  salt  solution  is  then  added  so  that  the  required  number 
of  previously  determined  haemolytic  units  shall  be  present  for  every  unit 
(1.0  c.c.  of  5  per  cent)  of  red  cells.  Therefore,  supposing  that  0.001  c.c. 
of  the  immune  serum  was  found  to  be  one  haemolytic  amboceptor  unit, 
1.0  c.c.  of  the  1-1000  dilution  of  this  serum  added  to  1.0  c.c.  of  a  5  per 
cent  emulsion  of  red  cells  would  sensitize  them  with  one  unit.  Having 
made  such  a  mixture  and  having  given  it  15  minutes  to  allow  the  union 
to  take  place,  every  2.0  c.c.  would  represent  1.0  c.c.  of  5  per  cent  red 
cells  and  1  unit  of  amboceptor  united.  Or  if  desired  the  cells  could  be 
centrifugalized,  washed,  and  made  up  to  the  original  concentration  of 
5  per  cent.  On  this  principle  any  number  of  units  up  to  the  maximum 
absorption  power  can  be  added  to  the  red  cells.  As  we  approach  the 
maximum  absorption  power  of  amboceptor  by  red  cells  it  is  always 
well  to  centrifugalize  and  reemulsify  the  red  cells  so  that  our  experi- 
ment may  not  be  confused  by  the  presence  of  excessive  unabsorbed 
amboceptor. 


42 


LABORATORY  COURSE  IN  SERUM  STUDY 


Each  student  needs: 
10  c.c.  of  5%  sheep  cells 
2  c.c.  amboceptor  (diluted  1-100) 
2  c.c.  complement  undiluted,  for  each  three  students 
1         test  tube  rack 
20         half-inch  test  tubes 
4         1-c.c.  pipettes,  and  flask  salt  solution 
4        f -inch  test  tubes 

LESSON  V 

QUANTITATIVE  RELATIONS  OF  AMBOCEPTOR  AND  COMPLE- 
MENT  (Continued) 

Experiment  1 

THE  ACTIVITY  OF  COMPLEMENT  DEPENDS  TO  SOME  EXTENT  ON  ITS 
CONCENTRATION  AND  NOT  ONLY  ON  THE  TOTAL  AMOUNT  PRESENT 

A  25  per  cent  suspension  of  washed  sheep  cells  is  made. 

A  series  of  tubes  is  set  up  (if  necessary  by  instructor)  in  which  the 
minimal  amount  of  amboceptor  necessary  to  lake  0.1  c.c.  of  these  cells 
in  the  presence  of  0.05  c.c.  of  undiluted  complement  and  in  a  total  volume 
of  1.0  c.c.  is  determined.  The  hsemolytic  serum  (amboceptor)  is  then 
diluted  so  that  this  minimal  amount  is  contained  in  0.1  c.c. 

The  student  then  mixes  2.0  c.c.  of  this  amboceptor  dilution  with 
2.0  c.c.  of  the  25  per  cent  red  cell  suspension.  He  then  determines,  in 
the  following  preliminary  titration,  the  minimal  amount  (unit)  of  com- 
plement which  will  lake  0.1  c.c.  of  these  cells  in  one  hour,  in  a  total 
volume  of  1  c.c. 


GUINEA-PIG  SERUM  1-10 

SENSITIZED  CELLS 
(.05  C.C.  CELLS  +  .05  C.C. 
OF  AMBOCEPTOR) 

SALINE  UP  TO  1.0  C.C. 

Tube  1 
Tube  2 
TubeS 
Tube  4 
TubeS 
Tube  6 
Tube? 
TubeS 

0.15  C.C. 
0.2 
0.25 
0.3 
0.4 
0.5 
0.0 
.5 

0.1  c.c. 
0.1  c.c. 
0.1  c.c. 
0.1  c.c. 
0.1  c.c. 
0.1  c.c. 
0.1 
.05  unsensi- 
tized  cells 

.75  C.C. 

.7 
.65 
.6 
.5 
.4 
.9 
.45 

Then  with  1,  5,  and  10  times  this  minimal  amount  in  the  tubes  as  indi- 
cated in  the  protocol  the  experiment  is  set  up.  The  protocol  given 
below  is  constructed  on  the  supposition  that,  in  the  above  titration,  0.2 
c.c.  of  a  1  to  10  dilution  of  complement  was  the  smallest  amount  of  the 
complement  which  gave  complete  laking. 


44 


LABORATORY  COURSE  IN  SERUM  STUDY 


GUINEA  Pio  SERUM 

1   IN   10 

SALT  SOLUTION  UP  TO 

RESULTING  DILUTION 
OF  GUINEA  PIG  SERUM 

Tube    1 

0.2  C.C. 

1.0  c.c. 

in    50 

Tube    2 

0.2 

2.0 

in  100 

Tube    3 

0.2 

5.0 

in  250 

Tube    4 

0.2 

10.0 

in  500 

Tube    5 

1.0 

1.0 

in    10 

Tube    6 

1.0 

5.0 

in    50 

Tube    7 

1.0 

10.0 

in  100 

Tube    8 

1.0 

20.0 

in  200 

Tube    9 

2.0 

5.0 

in    25 

Tube  10 

2.0 

10.0 

lin    50 

Tube  11 

2.0 

20.0 

1  in  100 

To  each  tube  add  1  unit  (0.1  c.c.)  of  cells  previously  sensitized  with 
1  unit  of  amboceptor. 

Calculate  the  concentration  of  complement  in  the  tubes  used  in  the 
preliminary  complement  titration,  and  compare  the  results.  If  the 
activity  of  complement  does  depend  upon  the  concentration,  complete 
laking  should  occur  in  one  hour  in  those  tubes  of  the  second  series 
which  contain  a  dilution  of  guinea  pig  serum  equal  to  that  in  the  first 
tube  of  the  first  series  which  showed  complete  haemolysis. 

Experiment  2 

THE  ACTIVITY  OF  SENSITIZER  OR  AMBOCEPTOR  DEPENDS  ON  ITS 
TOTAL  AMOUNT  AND  NOT  ON  ITS  CONCENTRATION 

Suspend  1  unit  (0.05  of  25%)  of  cells  in  varying  amounts  (1,  5,  10, 
20  c.c.)  of  salt  solution. 

To  each  tube  add  1  unit  of  amboceptor. 

Mix  and  keep  at  37°  C.  for  one  half  hour. 

Centrifugalize. 

To  the  sediment  in  each  tube  add  1  unit  of  complement,  as  previously 
determined,  and  sufficient  salt  solution  to  bring  the  volume  to  1  c.c. 

Incubate. 

All  tubes  should  show  complete  haemolysis  in  1  hour  if  the  cells 
have  absorbed  the  entire  unit  of  amboceptor. 

Each  student  needs: 

2  c.c.  of  25%  sheep  cells  1  ten-c.c.  pipette 

(about)    2  c.c.  of  undiluted  com-  2  test  tube  racks  for  12  J-inch 

plement  test  tubes 

25  units  of  amboceptor  20  J-inch  test  tubes 

Flask  of  saline  1  fifty-c.c.  centrifuge  tube 

4  one-c.c.  pipettes  3  fifteen-c.c.  centrifuge  tubes 

6  £-inch  test  tubes 


46          LABORATORY  COURSE  IN  SERUM  STUDY 

LESSON  VI 

THE  UNION  OF  IMMUNE-BODY   (AMBOCEPTOR  OR   SENSI- 

TIZER)   AND   CELLS 

\ 

1.   DISSOCIATION  OF  AMBOCEPTOR  (Mum) 

RED  cells  have  a  very  great  affinity  for  their  specific  immune- 
body.  They  can  absorb  a  great  number  of  units.  Nevertheless  at 
37°  C.  corpuscles  containing  multiple  doses  of  amboceptor  give  off 
a  certain  amount  to  the  surrounding  fluid  when  it  is  free  of  it. 
There  appears  to  be  an  equilibrium  between  combined  and  free  im- 
mune-body. This  can  be  illustrated  by  the  following  experiment : 

To  1.5  c.c.  of  a"  5  per  cent  emulsion  of  sheep  corpuscles  is  added 
inactivated  haemolytic  serum  so  that  there  shall  be  20  units  of  ambo- 
ceptor x  to  every  0.5  c.c.  of  cells.  (The  student  should  calculate  and 
make  dilutions  for  this  purpose  after  being  told  the  unit  of  the  hsemolytic 
serum  given  him.) 

This  mixture  is  allowed  to  stand  at  room  temperature  for  30  minutes. 

Centrifugalize  and  set  aside  the  supernatant  fluid.     This  is  Tube  (a). 

Wash  the  cells  obtained  in  the  sediment  of  the  preceding  centrif  uga- 
tion  three  times  and  set  aside  the  salt  solution  remaining  as  supernatant 
fluid  of  the  last  washing.  This  is  Tube  (6). 

Make  the  suspension  of  red  cells  up  to  the  original  volume  by  the 
addition  of  saline  and  incubate  this  at  42°  C.  for  one  hour.  Then  centrif- 
ugalize  and  set  aside  the  supernatant  fluid  of  this,  which  constitutes 
Tube  (c). 

Make  the  sediment  of  the  cells  obtained  in  the  preceding  up  to  1.5 
c.c.  Shake.  Remove  0.5  c.c.  and  add  to  it  1.0  c.c.  of  10  per  cent  fresh 
guinea  pig  complement.  This  is  Tube  (d). 

To  (a),  (6)  and  (c),  each,  add  0.5  c.c.  of  5  per  cent  sheep  corpuscles 
and  .5  c.c.  of  complement  (10  per  cent  fresh  guinea  pig  serum  in  salt 
solution). 

All  four  tubes  are  incubated  at  37°  C.  for  one  hour. 

Record  the  degree  of  laking  in  each  tube. 

Tube  (a)  will  show  how  much  of  the  20  units  of  amboceptor  failed  to 
be  absorbed.  The  exact  amount  could  be  determined  by  titration,  which, 
however,  would  needlessly  prolong  the  experiment  and  add  nothing 
to  the  illustration  of  the  principle. 

1  The  unit  here  is  the  minimal  amount  which  lakes  0.5  c.c.,  not  1  c.c.,  of  cells. 
If  Experiments  1  and  3  of  this  lesson  are  to  be  done  on  the  same  afternoon  it  is 
well  to  sensitize  a  considerable  amount  of  cells  at  one  time. 


48 


LABORATORY   COURSE    IN   SERUM   STUDY 


Tube  (6)  will  show  whether  the  three  washings  were  sufficient  to 
remove  all  amboceptor  from  the  fluid  bathing  the  cells.  Any  excess  of 
amboceptor  remaining  unattached  to  the  cells  after  the  washing  would 
show  in  the  supernatant  fluid  by  laking. 

Tube  (c)  will  show  how  much,  if  any,  amboceptor  was  dissociated 
after  one  hour  at  37°  C. 


2.  VELOCITY  OF  AMBOCEPTOR  ABSORPTION 

Red  cells  absorb  their  homologous  immune-body  very  rapidly. 
A  knowledge  of  this  is  of  great  importance  as  the  following  ex- 
periments show. 

A 

Take  3  c.c.  of  5  per  cent  sheep  corpuscles  in  a  wide  test  tube  and  add 
3  c.c.  of  diluted  amboceptor  drop  by  drop,  shaking  constantly  (the 
dilution  of  the  amboceptor  is  such  that  1  unit  is  contained  in  0.5  c.c. ; 
the  unit  here  is  taken  as  the  minimal  amount  which  lakes  0.5  c.c.  of 
ceUs). 

B 

Take  3  c.c.  of  diluted  amboceptor  and  add  3  c.c.  of  5  per  cent  sheep 
cells  drop  by  drop,  shaking  constantly. 

Set  up  two  parallel  series  of  five  tubes  each  containing  varying 
amounts  of  guinea  pig  serum  (1-10). 

Series  1 


GUINEA  Pro  SERUM 

SALT  SOLUTION  (TO 

TUBE 

DILUTED  1-10 

MAKE  ALL  VOLUMES 

MIXTURE  A 

(COMPLEMENT) 

EQUAL) 

1 

0.75  c.c. 

0.0     C.C. 

1.0  c.c. 

2 

0.5    c.c. 

0.25  c.c. 

.  1.0  c.c. 

3 

0.4    c.c. 

0.35  c.c. 

1.0  c.c. 

4 

0.3    c.c. 

0.45  c.c. 

1.0  c.c. 

5 

0.2    c.c. 

0.55  c.c. 

1.0  c.c. 

Series  2 

Duplicate  above  with  mixture  B. 

Incubate  one  hour  and  compare  results. 

The  observed  differences  are  probably  explained  by  the  fact  that 
the  first  cells  which  are  added  to  B  absorb  nearly  all  the  amboceptor, 
leaving  insufficient  to  sensitize  the  last  cells  added. 


50  LABORATORY  COURSE  IN  SERUM  STUDY 

3.   LIBERATION  OF  AMBOCEPTOR  IN  HAEMOLYSIS 

When  red  cells  containing  several  hsemolytic  doses  of  ambo- 
ceptor  are  laked  by  a  not  excessive  amount  of  complement,  some 
of  the  amboceptor  is  liberated  in  the  solution. 

To  2.0  c.c.  of  5  per  cent  sheep  cells  add  sufficient  amboceptor  so 
that  there  will  be  20  units  for  every  unit  (0.5  c.c.)  of  5  per  cent  sheep 
cells.  Allow  to  stand  for  one  half  hour.  Centrifuge  and  wash  with 
saline  three  times  and  after  the  third  washing  make  the  volume  again 
up  to  2.0  c.c. 

To  1.0  c.c.  of  cells  so  sensitized  add  1.0  c.c.  of  10  per  cent  guinea-pig 
complement.  Incubate  one  half  hour.  Then  divide  into  two  equal 
portions  of  1.0  c.c.  each.  To  the  first  portion  add  0.5  c.c.  of  the  sen- 
sitized cells  and  incubate  again  for  one  hour.  Note  whether  haemolysis 
of  the  added  cells  occurs.  To  the  second  portion  add  0.5  c.c.  of  un- 
treated corpuscle  suspension  and  1.0  c.c.  of  10  per  cent  complement. 
Haemolysis,  after  incubation  at  37J  degrees  centigrade,  will  indicate 
that  amboceptor  was  liberated  by  the  laking  of  the  sensitized  cells  dur- 
ing the  first  incubation  and  was  taken  up  by  the  added  cells  in  the  second 
incubation. 

How  does  the  amount  of  amboceptor  liberated  compare  with  the 
amount  dissociated  by  simple  incubation  without  haemolysis  in  Experi- 
ment 1  ? 

Each  student  needs : 

12  c.c.  of  5%  sheep  cells 
160  units  of  amboceptor 
10  c.c.  of  complement  (1-10) 
Flask  saline 

4  one-c.c.  pipettes 

1  test  tube  rack  for  half-inch  test  tubes 

1  graduated  centrifuge  tube 
20  half-inch  test  tubes 

3  three-quarter-inch  test  tubes 

1  five-c.c.  pipette 

1  nipple  pipette1 

1  If  experiments  1  and  3  of  this  lesson  are  to  be  done  on  the  same  afternoon,  it 
is  well  to  sensitize  a  considerable  amount  of  cells  at  one  time. 


52          LABORATORY  COURSE  IN  SERUM  STUDY 

LESSON  VII 
PFEIFFER  PHENOMENON 

VIRULENT  cholera  spirilla  injected  into  the  peritoneal  cavity 
of  a  normal  guinea  pig  proceed  to  multiply  rapidly  after  a  pe- 
riod of  about  half  an  hour,  and  the  animal  dies  usually  within  24 
hours  with  symptoms  of  profound  intoxication.  The  same  spirilla 
injected  into  an  animal  which  has  been  immunized  by  previous 
injections  of  killed  spirilla  or  of  sub-lethal  doses  of  the  living 
microorganisms  become  granular  and  in  some  cases  swell,  take 
on  globular  and  vacuolated  forms  and  gradually  undergo  lysis. 
The  guinea  pig  in  such  cases  recovers  completely  from  the  in- 
jection. This  destruction  of  invading  bacteria  by  the  immune 
animal  was  termed  by  Pfeiffer  " bacteriolysis."  It  has  been  ob- 
served with  certain  of  the  Gram-negative  bacilli  such  as  the 
typhoid  and  the  dysentery  bacillus,  but  is  best  observed  in 
cholera.  Similar  lysis  is  observed  if  the  spirilla  are  injected  into 
a  normal  pig  together  with  serum  of  an  immunized  guinea  pig  or 
rabbit. 

For  this  experiment  it  is  necessary  to  employ  a  virulent 1  strain 
of  cholera  spirilla,  since  old  laboratory  cultures  do  not  show  the 
phenomenon  sharply  and  rarely  kill  the  guinea  pigs  in  the  con- 
trols. 

The  lethal  dose  of  the  particular  strain  of  cholera  used  for 
the  guinea  pigs  should  be  determined  roughly  before  carrying  out 
the  experiment. 

It  is  inadvisable  to  allow  students,  except  in  very  small  groups, 
to  handle  virulent  cholera  cultures,  so  that  in  carrying  out  this 
experiment,  the  preparation  of  the  cholera  suspension,  the  in- 
jection of  the  pig  and  the  removal  of  specimens  from  the  peri- 
toneum, as  described  below,  should  be  carried  out  by  the  instruc- 
tor. The  smears  should  be  fixed  immediately  in  10  per  cent 
foimalin,  after  which  they  may  be  distributed  to  the  members  of 
the  class  for  study. 

Two  pigs  —  one  normal,  the  other  previously  immunized  with  cholera 
spirilla  —  are  intraperitoneally  injected  with  the  determined  dose,  say  a 

1  The  strain  must  be  recently  isolated  or  one  that  has  been  passed  through  guinea 
Digs  until  it  will  kill  in  quantities  of  one  loopful  of  an  agar  slant. 


54          LABORATORY  COURSE  IN  SERUM  STUDY 

twentieth  of  the  emulsion  obtained  from  an  agar  slant,  and  after  ten 
minutes  a  specimen  of  the  peritoneal  fluid  is  withdrawn  for  observation. 
This  is  done  by  shaving  the  left  side  of  the  abdomen  of  the  pig,  holding 
the  pig,  back  down,  on  a  tray.  This  can  be  done  satisfactorily  with  the 
operator's  left  hand.  The  shaved  surface  of  the  skin  is  sterilized  by 
wiping  with  5  per  cent  carbolic,  a  small  cut  made  through  the  skin, 
but  not  through  the  muscles,  with  a  sharp  pair  of  scissors.  Then  the 
sharp  tip  of  a  capillary  pipette  is  inserted  through  the  muscles  and  the 
peritoneum.  If  the  pipette  is  held  horizontally  or  with  the  open  end 
depressed,  a  small  amount  of  peritoneal  fluid  will  run  up  into  the  capillary 
portion  even  if  no  suction  is  used.  When  sufficient  amount  of  fluid 
[0.1  or  0.2  c.c.]  is  obtained,  the  pipette  is  withdrawn  and  the  puncture 
covered  with  cotton  soaked  in  carbolic  solution. 

A  hang-drop  preparation  is  made  with  a  drop  of  this  fluid  and  the 
remainder  blown  out  into  a  watch  glass.  With  a  platinum  loop  a  series 
of  thin  smears  are  made  of  this  fluid  on  glass  slides,  allowing  one  slide 
for  each  member  of  the  class,  and  as  soon  as  the  films  are  dried  they  are 
placed  in  Coplin  jars  containing  a  10  per  cent  solution  of  formalin 
(4  per  cent  formaldehyde)  for  five  minutes.  They  are  then  withdrawn, 
allowed  to  dry,  and  are  stained  by  the  students  with  methylene  blue  and 
studied  under  the  oil  immersion  lens.  The  hang-drop  preparation 
should  be  observed  under  a  high-power  dry  lens  and  one  preparation 
may  be  observed  by  the  whole  class. 

Preparations  are  made  in  this  way  from  the  normal  and  immune  pig 
ten  minutes,  thirty  minutes,  one  hour  and  two  hours  respectively  after 
injection.  By  the  end  of  this  period  if  a  proper  dose  of  the  organisms 
has  been  injected,  the  spirilla  will  be  found  to  have  multiplied  enor- 
mously in  the  peritoneal  fluid  of  the  normal  animal,  whereas  in  the 
smears  made  from  the  immune  animal  after  thirty  minutes  to  an  hour 
the  spirilla  will  appear  granular  or  swollen,  and  after  two  hours,  or,  if 
the  observation  is  followed  that  long,  after  three  or  four  hours,  will  be 
found  to  have  practically  disappeared. 

The  two  animals  should  be  kept  for  observation  after  the  experiment, 
and  the  unprotected  animal  will  probably  be  found  dead  the  next  day. 

The  passive  transference  of  bacteriolytic  immunity  to  another 
animal  by  simultaneous  injection  of  cholera  spirilla  and  anti-cholera 
serum  obtained  from  the  rabbit  immunized  to  cholera  may  be  demon- 
strated in  a  similar  way,  and  by  this  technic  the  strength  of  a  bacteriolytic 
serum  may  be  titrated  by  injecting  series  of  guinea  pigs  with  varying 
amounts  of  the  serum. 


56          LABORATORY  COURSE  IN  SERUM  STUDY 

A  type  protocol  of  such  an  experiment  is  given  below: 


WEIGHT  or 
GUINEA  Pio 

DOSE  OF 
BACTERIA1 
CHOLEBA 
SPIRILLA 

AMOUNT  OP 
INACTIVATED 
IMMUNE  SERUM 

RESULT 

(1)  215  gm. 

2  mg. 

0.1  c.c.  in  1  c.c. 

Complete   dissolution    in   less 

salt  solution 

than  one  hour.     Lives. 

(2)  230  gm. 

2  ing. 

0.05  c.c. 

About  the  same  as  first. 

(3)  200  gm. 

2  ing. 

0.01  c.c. 

Somewhat  slower  than  in  other 

two  ;   a  few  unchanged  spirilla 

after  1  hour.     Final  dissolution. 

Pig  lives. 

(4)  245  gm. 

2  mg. 

0.005  c.c. 

Similar   to   (3)    but   complete 

dissolution  in  2  hours.     Pig  lives. 

(5)  220  gm. 

2  mg. 

0.001  c.c. 

After  30  minutes  the  spirilla 

seem  to  have  begun  to  multiply. 

Dies    with    innumerable    active 

spirilla  in  peritoneum. 

Normal  Con- 

trol 

(6)  210  gm. 

2  mg. 

0.1  c.c.  normal 

Very  slight  lysis  at  the  begin- 

inactive rab- 

ning.   Soon  rapid  multiplication. 

bit  serum 

Dies. 

LESSON  VIII 
BACTERICIDAL  TEST  IN  VITRO  —  STERN    KORTE 

THE  destruction  of  bacteria  which  takes  place  in  the  peri- 
toneum of  immune  animals  may  also  be  demonstrated  for  certain 
species  of  bacteria  in  the  test  tube.  Small  quantities  of  typhoid 
bacilli,  as  shown  in  a  previous  experiment,  are  killed  by  exposure  to 
certain  normal  sera  such  as  those  of  the  rabbit  and  the  guinea  pig. 
A  more  powerful  action  is  observed  in  sera  of  animals  immunized 
to  the  typhoid  bacillus,  and,  as  in  the  case  of  red  cells,  the  immune 
body  or  sensitizer  can  be  shown  to  be  relatively  thermostable  in 
that  it  will  act  after  exposure  to  56°  C.  for  half  an  hour  provided  it 
is  reactivated  by  the  addition  of  a  small  amount  of  normal  serum. 

The  bactericidal  effect  of  immune  serum  shows  more  clearly 
than  most  other  antibody  phenomena  the  presence  of  a  prezone, 

1  The  bacteria  may  be  measured  for  such  an  experiment  by  standard  loop- 
fuls  (one  loop  being  equal  to  2  milligrams),  or  by  volume  in  emulsion  with  salt 
solution. 


58 


LABORATORY   COURSE    IN   SERUM   STUDY 


that  is,  the  failure  of  the  antibodies  to  act  if  present  in  excessive 
concentration.  In  the  sera  of  artificially  immunized  animals  or  of 
typhoid  patients  amounts  greater  than  0.01  c.c.  of  a  strongly  im- 
mune serum  are  usually  ineffective,  the  best  results  being  obtained 
with  amounts  varying  from  0.001  to  0.0001  c.c.  in  a  volume  of  1  c.c. 

Since  the  inactivated  immune  serum  must,  of  course,  be  re- 
activated by  fresh  normal  serum  used  as  complement  or  alexin, 
it  is  important  to  use  an  amount  of  the  normal  serum  too  small  to 
be  of  itself  bactericidal ;  and  the  first  step  to  determine  is  the 
bactericidal  power  of  the  normal  serum  against  the  strain  of 
bacteria  to  be  used.  On  the  other  hand,  to  secure  striking  results 
it  is  necessary  to  use  as  large  an  amount  of  fresh  normal  serum 
as  possible  without  entering  the  zone  of  normal  bactericidal 
power.  Normal  rabbit  serum  is  usually  definitely  bactericidal 
for  typhoid  bacilli  in  amounts  ranging  from  0.1  to  0.02  c.c.  (in  a 
total  volume  of  1  c.c.). 

Reagents : 

1.  Anti-typhoid  serum  —  serum  of  a  rabbit  immunized  against 
typhoid  bacilli  obtained  in  sterile  condition  and  heated  at  56°  C.  for  half 
an  hour. 

2.  Normal  rabbit  serum,  which  must  also  be  sterile. 

3.  24-hour  broth  culture  of  typhoid  bacilli. 

The  glassware  and  salt  solution  used  in  the  experiment  must,  of 
course,  be  sterile,  and  the  reagents  handled  with  care  to  avoid  contam- 
ination during  the  setting  up  of  the  experiment. 

TENTATIVE  SCHEDULE  l 
Set  up  the  following  tubes: 


IMMUNE  SERUM 

TUBE 

Actual  Quantity  of  the 

SOLUTION 

SERUM  1-50 

Immune  Serum 

1 

0.01  C.C. 

0.5    c.c.  (1-50) 

0.0    c.c. 

0.5  c.c. 

2 

0.002  c.c. 

0.1    c.c.  (1-50) 

0.4    c.c. 

0.5  c.c. 

a 

0.001  c.c. 

0.5    c.c.  (1-500) 

0.0    c.c. 

0.5  c.c. 

4 

0.0001  c.c. 

0.05  c.c.  (1-500) 

0.45  c.c. 

0.5  c.c. 

5 

*  0.01  c.c. 

0.5    c.c.  (1-50) 

0.5    c.c. 

— 

6 

None 

—             — 

0.5    c.c. 

0.5  c.c. 

7 

None 

—            — 

1.0    c.c. 

— 

8 

None 

—            — 

1.0    c.c. 

— 

1  By  a  previous  test  of  the  serum  the  instructor  should  adjust  the  schedule  so 
that  one  or  two  tubes  lie  within  the  "killing  "  zone  and  others  above  and  below  it. 

2  The  serum  in  this  control  tube  should  be  that  amount  which  shows  maximum 
bactericidal  power  when  used  with  active  serum. 


60  LABORATORY  COURSE  IN  SERUM  STUDY 

Add  to  each  tube  about  0.1  c.c.  of  a  24  hour  broth  culture  of  B. 
typhosus,  diluted  1-500.  This  should  be  measured  by  making  a  mark 
on  a  sterile  capillary  pipette,  filling  accurately  to  this  mark  and  dis- 
charging the  contents  against  the  side  of  the  tube,  close  to  the  level  of 
the  fluid,  being  careful  not  to  touch  the  upper  portion.  The  actual 
amount  used  is  unimportant,  but  precisely  equal  amounts  must  be  added 
to  each  tube.  Shake.  Incubate  the  tubes  two  hours  at  37°  C.  At  the 
end  of  this  time  convey  two  loopsful  from  each  tube  into  melted  and  cooled 
agar  tubes  and  pour  plates  in  the  usual  way.  Transfer  of  two  loops  from 
tube  eight  should  also  be  made  into  agar  in  this  way  immediately  after 
mixing  without  incubating. 

In  careful  experiments,  two  additional  controls,  the  one  containing 
0.5  c.c.  of  1-50  normal  serum,  the  other  containing  0.5  c.c.  of  1-50  im- 
mune serum  without  the  addition  of  typhoid  bacilli,  should  also  be  set 
up  and  transfers  should  be  made  from  them  into  agar  to  prove  the  sterility 
of  the  reagents  used.  A  successful  experiment  should  show  an  enormous 
number  of  colonies,  over  10,000  to  the  plate,  in  plates  Nos.  5,  6  and  7. 
Plate  8  should  also  show  a  large  number  of  colonies,  though  perhaps  less 
than  the  other  controls.  In  plates  2  and  3  there  should  be  very  few  col- 
onies or  none  at  all;  in  plates  1  and  4  some  reduction  in  the  number, 
though  in  some  cases  there  may  be  no  observable  difference  between 
these  plates  and  the  controls. 

Each  student  needs:  Five  f-inch  test  tubes,  twelve  petri  dishes, 
twelve  tubes  of  agar,  four  one-c.c.  pipettes. 

LESSON  IX 
I.  TITRATION  OF  AGGLUTININS  BY  MACROSCOPIC  METHOD 

THE  clumping  of  bacteria  by  immune  serum  causes  a  homo- 
geneous suspension  to  form  flocculi  easily  visible  to  the  naked  eye, 
which,  on  standing,  settle  to  the  bottom  of  the  tube,  making 
the  supernatant  fluid  clear.  The  macroscopic  test  is  considered 
more  accurate  than  the  microscopic  since  one  is  less  often  de- 
ceived by  small  clumps  of  bacteria  which  may  form  spontaneously 
in  salt  solution  or  broth  for  various  reasons  and  are  readily 
visible  under  the  microscope  but  do  not  come  together  in  large 
enough  masses  to  cause  a  precipitate  visible  to  the  naked  eye.  It 
is  much  simpler  to  carry  out  a  quantitative  titration  of  the  aggluti- 
native power  of  the  serum  by  setting  it  up  in  test  tubes  than 
by  preparing  hang-drops  of  each  dilution.  Furthermore,  since 
the  quantities  used  in  these  tests  are  relatively  large,  slight  inac- 
curacies in  measurement  do  not  lead  to  such  gross  errors  as  would 


62          LABORATORY  COURSE  IN  SERUM  STUDY 

similar  inaccuracies  in  the  microscopic  method.  For  these 
reasons  the  macroscopic  method  is  the  one  usually  employed  in 
experimental  investigations.  On  the  other  hand,  in  testing 
patient's  sera  against  the  typhoid  bacillus  (the  Widal  reaction) 
considerable  information  is  given  by  the  cessation  of  motility  of 
the  bacilli  in  the  presence  of  immune  serum.  It  is  desirable  to 
use  small  amounts  of  serum  and  unnecessary  to  set  up  more  than 
three  or  four  dilutions  in  one  test. 

The  macroscopic  test  may  be  carried  out  with  living  bacteria, 
with  bacteria  killed  either  by  exposure  to  heat  (56°  C.)  or  by  ex- 
posure to  weak  antiseptics  such  as  phenol,  1  per  cent  solution,  or 
formaldehyde  0.8  per  cent.  Killed  suspensions  of  bacteria  may 
be  kept  a  long  time  in  the  ice  box  and  still  remain  serviceable. 
The  flocculation  is  most  easily  observed  in  narrow  test  tubes, 
f  of  an  inch  in  diameter,  but  larger  tubes  can  also  be  used  with- 
out difficulty.  Some  workers  prefer  to  use  tubes  pointed  at  the 
bottom,  similar  to  the  centrifuge  tubes,  since  the  sediments 
which  settle  out  can  be  readily  observed  in  the  tips  of  these  tubes. 

The  period  of  incubation  varies.  A  marked  reaction  is  visible 
after  an  hour  at  37°,  but  with  many  organisms  it  is  necessary  to 
incubate  for  two  hours  to  obtain  clear-cut  results,  and  the  floc- 
culation often  becomes  more  distinct  when  the  tubes  are  removed 
from  the  water  bath  and  placed  in  the  ice  box  for  from  half  an 
hour  to  12  hours.  Such  organisms  as  the  streptococcus1  and  the 
staphylococcus  usually  require  the  longer  periods  of  incuba- 
tion, and  agglutination  is  favored  by  a  temperature  of  56°.  On 
the  other  hand,  for  organisms  of  the  typhoid-colon  group  long  in- 
cubation is  unnecessary. 

Reagents : 

1.  Serum  of  a  rabbit  immunized  against  typhoid.    For  this  test  the 
serum  of  animals  immunized  by  the  class  should  be  used,  the  members 
in  charge  of  the  rabbits  bleeding  them  from  the  carotid  at  the  previous 
exercises  and  distributing  to  each  other  member  of  the  class  2  c.c.  of  a 
1-10  dilution  of  the  serum. 

2.  B.  typhosus  —  24  hour  culture  on  agar  slant. 

3.  Normal  salt  solution. 

1  Streptococcus  agglutination  requires  a  special  and  complic.it c<l  technique  to 
be  satisfactory  and  then  isn't. 


64          LABORATORY  COURSE  IN  SERUM  STUDY 


Prepare  a  suspension  of  typhoid  bacilli  by  adding  10  c.c.  of  salt 
solution  to  an  agar  slant  culture  and  gently  rubbing  the  growth  from 
the  surface  of  the  agar  by  means  of  a  platinum  loop  or  a  capillary  glass 
pipette,  being  careful  not  to  cut  the  surface  of  the  agar.  Shake  the  tube 
gently  without  wetting  the  stopper  and  allow  to  stand  for  10  minutes 
so  that  the  larger  clumps  may  settle  to  the  bottom.  Take  a  capillary 
glass  pipette,  plug  it  at  the  suction  end  with  non-absorbent  cotton  and 
provide  it  with  a  rubber  nipple.  Draw  off  8  or  9  c.c.  of  the  upper  por- 
tion of  the  suspension,  being  careful  not  to  disturb  the  sediment  and 
place  in  a  separate  test  tube,  taking  care  that  the  suspension  does  not 
flow  against  the  upper  portions  of  the  tube.  Add  0.2  c.c.  of  40  per  cent 
formaldehyde  and  place  in  water  bath  at  56°  for  thirty  minutes.  If 
clumps  are  still  visible  the  suspension  may  be  filtered  through  a  moist 
paper. 

B 

Prepare  the  following  dilutions  of  the  immune  serum  in  test  tubes: 1 

1.  1-10      =  dilution  as  distributed 

2.  1-50      =  0.5  c.c.  of  1-10      dilution  +  2  c.c.  salt  solution 

3.  1-250    =  0.5  c.c.  of  1-50      dilution  +  2  c.c.  salt  solution 

4.  1-500    =1.0  c.c.  of  1-250    dilution  +  1  c.c.  salt  solution 

5.  1-1000  =1.0  c.c.  of  1-500    dilution  +  1  c.c.  salt  solution 

6.  1-2000  =  1.0  c.c.  of  1-1000  dilution  +  1  c.c.  salt  solution 


Place  0.5  c.c.  of  each  dilution  of  serum  (1-10  to  1-2000)  in  a  series  of 
agglutination  tubes  and  in  an  additional  tube  0.5  c.c.  of  salt  solution  for 
control. 

To  each  tube  add  0.5  c.c.  of  the  killed  typhoid  suspension.  This, 
of  course,  doubles  the  dilution  in  each  case  and  the  tubes  should  be 
labeled  1-20,  1-100,  1-500,  etc.,  up  to  1^000.  Shake  gently  till  the 
two  fluids  mix.  The  typhoid  suspension  should  be  distributed  by  means 
of  a  pipette  carefully  plugged  at  the  upper  end  with  non-absorbent 
cotton.  This  should  be  used  wherever  bacteria  are  handled  with  a 
pipette,  even  when  they  are  thought  to  have  been  killed. 

Place  the  tubes  in  the  water  bath,  at  37°  C.,  for  one  hour,  and 
observe  carefully  by  transmitted  light.  Record  the  results  and  place 
the  tubes  in  the  ice  box  for  one  half  hour  and  again  make  a  reading. 
Read  again  on  the  following  day. 

1  Instructor  should  adjust  this  schedule  by  a  previous  titration. 


66 


LABORATORY  COURSE  IN  SERUM  STUDY 


TUBE 

SERUM 

TYPHOID  EMULSION 

RESULT 

1 

1-10      0.5  c.c. 

0.5  c.c. 

2 

1-50      0.5  c.c. 

0.5  c.c. 

3 

1-100    0.5  c.c. 

0.5  c.c. 

4 

1-250    0.5  c.c. 

0.5  c.c. 

5 

1-500    0.5  c.c. 

0.5  c.c. 

6 

1-1000  0.5  c.c. 

0.5  c.c. 

7 

Salt  sol.                      0.5  c.c. 

0.5  c.c. 

8 

Normal  serum  1-20  0.5  c.c. 

0.5  c.c. 

D 

The  students  in  charge  of  the  immune  rabbits  should  test  at  the  same 
time  the  samples  of  serum  which  were  obtained  from  the  rabbits  before 
they  received  their  first  injection  and  which  have  been  stored  in  the 
ice  box  meanwhile.  In  this  case  one  tube  of  undiluted  serum  should  be 
tested,  one  of  a  1-10  dilution,  one  of  1-50.  It  is  not  necessary  to  test 
higher  dilutions. 

H.    MACROSCOPIC  METHOD   OF  PROSCHER 

Take  four  glass  saltcellars.  In  the  first  place  0.5  c.c.  of  1-10  dilu- 
tion of  serum  used  in  the  first  experiment ;  in  the  second  0.5  c.c.  of  1-500 
serum ;  and  in  the  third  0.5  c.c.  of  1-2000  dilution ;  in  the  fourth  0.5  c.c. 
of  salt  solution.  To  each  add  0.5  c.c.  of  typhoid  suspension.  The  salt- 
cellars are  stacked  one  on  top  of  the  other  and  placed  in  the  incubator 
for  one  hour.  At  the  end  of  this  time  they  are  taken  out  and  may  be 
studied  under  low  power  of  the  microscope.  A  record  is  made  of  the 
amount  of  clumping  in  each  preparation. 

m.     THERMOSTABILITY   OF  AGGLUTININS 

The  agglutinins  like  the  bactericidal  sensitizing  substance  are 
resistant  to  moderate  heating  but  they  do  not  require  the  reacti- 
vation by  the  heat-sensitive  alexin  which  is  present  in  normal 
serum.  They  are,  however,  destroyed  by  exposure  to  tem- 
peratures of  from  70°  to  80°,  in  which  respect  they  also  resemble 
the  sensitizing  substance. 

In  each  of  three  agglutination  tubes  place  0.5  c.c.  of  a  1-10  dilution 
of  the  immune  serum.  Place  one  tube  in  a  water  bath  at  56°  C.  for  one 
half  hour.  Place  a  second  tube  in  a  water  bath  at  73°  C.  for  one  half 
hour.  Reserve  the  third  tube  as  a  control.  After  heating  add  0.5  c.c.  of 


68  LABORATORY    COURSE    IN    SERUM    STUDY 

the  killed  typhoid  suspension  to  each  tube  and  incubate  as  in  the  first 
experiment.  It  should  be  noted  that  the  sensitiveness  of  the  immune 
bodies  to  heat  varies  somewhat  according  to  the  dilution  in  which  they 
are  exposed,  and  if  desired,  exposure  to  56°  C.  may  be  repeated  with  un- 
diluted serum. 

The  student  is  referred  to  the  literature  given  in  the  text-book  on 
the  work  of  Joos  and  others  on  the  variation  in  heat  stability  of  agglu- 
tinins  according  to  whether  they  are  produced  by  the  injection  of  heated 
or  unheated  bacteria. 

Each  student  needs:  2  c.c.  typhoid  immune  serum,  1  typhoid 
culture  on  agar  slant. 

Normal  saline,  8  f-inch  test  tubes,  2  1-c.c.  pipettes,  12  f-inch  test 
tubes,  4  salt  cellars. 

LESSON  X 

AGGLUININS  (Continued) 

I.    AGGLUTINATION  BY  MICROSCOPIC  METHOD 
(WIDAL  TEST) 

DILUTIONS  for  the  microscopic  test  may  be  made  in  the  same 
manner  as  those  for  the  macroscopic,  but  it  is  often  desirable  in 
clinical  tests  to  work  with  smaller  amounts  of  serum  than  can  be 
measured  accurately  in  the  ordinary  graduated  pipettes.  In  this 
case  capillary  pipettes  are  used  and  the  serum  is  measured  either 
by  counting  drops  of  serum  and  of  salt  solution,  the  same  pipette 
held  at  the  same  angle  being  used  for  both  to  insure  accuracy, 
or  by  using  as  an  arbitrary  unit  the  amount  of  fluid  contained  in 
a  capillary  pipette  from  the  tip  to  a  wax  pencil  mark  placed  about 
an  inch  from  the  tip.  Students  should  practice  making  dilu- 
tions by  both  of  these  methods.  The  former  is  the  usual  method 
employed  in  diagnostic  work ;  the  latter,  however,  is  more  ac- 
curate. 

Reagents : 

1.  Serum  of  rabbit  immunized  to  B.  typhosus. 

2.  B.  typhosus  —  24  hour  culture  in  broth. 

3.  Salt  solution. 

A 

Take  four  watch  glasses  or  saltcellars  and  by  means  of  a  capillary 
pipette  provided  with  a  rubber  nipple  place  9  drops  of  salt  solution  in  the 
first  watch  glass  and  5  drops  in  each  of  the  other  three.  With  the  same 
pipette  place  one  drop  of  serum  in  the  first  saltcellar;  (if  a  highly 


70 


LABORATORY  COURSE  IN  SERUM  STUDY 


immune  animal  serum  is  used  this  should  previously  be  diluted  about 
1-100).  Mix  this  dilution  of  serum  thoroughly  by  drawing  it  in  and  out 
of  the  pipette  several  times.  Then  place  5  drops  of  this  first  dilution 
in  the  second  watch  glass.  Mix  the  contents  of  the  second  glass  as 
before.  Place  5  drops  from  the  second  glass  in  the  third  glass ;  return 
the  remainder  of  the  fluid  to  the  second  glass,  mix  the  contents  of  the 
third  watch  glass  and  remove  5  drops  of  this  last  to  make  its  volume 
equal  to  that  of  the  others.  We  now  have  four  watch  glasses,  each  con- 
taining 5  drops  of  fluid,  representing  1-10,  1-20,  1^0  dilutions  of  the 
serum  in  the  first  three,  and  salt  solution  in  the  fourth.  The  pipette  is 
now  thoroughly  rinsed  with  salt  solution  and  filled  with  the  typhoid 
broth.  Five  drops  of  the  broth  are  allowed  to  fall  into  each  of  the 
watch  glasses,  reducing  the  dilutions  to  1-20,  1^0,  and  1-80.  These  are 
the  usual  dilutions  used  in  the  Widal  test  for  diagnosis.  By  means 
of  a  platinum  loop  a  series  of  hang-drop  preparations  is  then  made, 
first  with  the  control,  then  of  the  highest  dilution,  etc.  The  drops  are 
allowed  to  stand  at  room  temperature  for  an  hour  and  are  then  studied 
under  a  high,  dry  lens  of  a  microscope  and  the  completeness  of  the  clump- 
ing and  the  loss  of  motility  in  the  different  preparations  recorded. 


WATCH  GLASS  No.  1 

WATCH  GLASS  No.  2 

WATCH  GLASS  No.  3 

WATCH  GLASS  No.  4 

Salt  solution 
9  drops 

Salt  solution 
5  drops 

Salt  solution 
5  drops 

Salt  solution 
5  drops 

Immune  serum 
1  drop 

Mixture  No.  1 
5  drops 

Mixture  No.  2 
5  drops 

0 

Discard  5  drops  of  Mixture  No.  3  and  add  5  drops  of  a  broth  culture 
of  B.  typhosus  to  each  glass. 

B 

A  similar  test,  using  the  same  reagents,  measured  by  the  Wright 
technique,  should  also  be  set  up : 

Take  four  watch  glasses  as  before  and  a  capillary  pipette,  broken  off, 
squarely  tipped,  stoppered  with  cotton,  and  provided  with  a  rubber 
nipple.  Place  a  mark  with  a  wax  pencil  about  one  inch  from  the  tip  of 
the  pipette.  Pour  out  salt  solution  into  a  watch  glass  or  cover  of  a 
Petri  dish  so  that  it  is  easily  reached  with  a  pipette.  Draw  up  salt  solu- 
tion to  the  mark,  admit  a  bubble  of  air,  draw  up  salt  solution  again  to  the 
mark,  admit  a  bubble  of  air,  and  proceed  until  9  units  of  salt  solution  have 
been  taken  up.  Then  take  up  serum  to  the  mark  and  discharge  the 


72  LABORATORY   COURSE   IN   SERUM   STUDY 

entire  contents  of  the  pipette  into  the  first  watch  glass ;  mix  by  drawing 
the  solution  in  and  out  of  the  pipette  several  times.  Take  up  five  units 
of  salt  solution  into  the  pipette  in  similar  manner,  then  five  units  of  the 
first  dilution,  and  place  this  mixture  in  the  second  watch  glass.  Place 
five  units  of  salt  solution  and  five  units  of  the  second  dilution  in  the  third 
watch  glass.  Remove  five  units  from  the  third  watch  glass  and  discard. 
Place  five  units  of  salt  solution  alone  in  the  fourth  glass.  Then  with  this 
same  pipette  measure  five  units  of  typhoid  broth  into  each  of  the  watch 
glasses.  Prepare  hang-drops  and  incubate  and  observe  as  above. 

Another  method  commonly  used  is  to  prepare  the  dilutions  of  serum 
in  one  of  the  two  ways  given  above,  and  then  place  on  a  series  of  cover 
glasses  with  a  platinum  loop  a  drop  of  typhoid  broth,  then  on  each  glass 
with  the  same  loop  a  drop  of  diluted  serum,  after  which  the  two  drops 
are  stirred  together  by  means  of  a  capillary  pipette  sealed  at  the  end. 
This  method  offers  no  advantages  over  the  preceding. 

The  method  sometimes  used  for  clinical  diagnosis,  of  diluting  with 
counted  loopfuls  of  saline  drops  of  dried  blood  sent  to  the  laboratory 
on  glass  slides,  is  exceeding  inaccurate. 

• 

II.  EFFECT  OF  SALT  ON  AGGLUTINATION 

The  agglutination  of  bacteria  which  occurs  in  the  presence  of 
immune  serum  has  many  analogies  to  the  flocculation  of  colloidal 
suspensions  by  electrolytes  and  is  dependent  on  the  presence  of 
electrolytes  in  the  solution.  Bacteria  sensitized  with  immunized 
serum  agglutinate  in  the  presence  of  traces  of  sodium  chloride 
and  other  inorganic  salts,  but  not  if  suspended  in  distilled  water. 
These  salts  may  also  cause  unsensitized  bacteria  to  agglutinate, 
and  false  clumping  due  to  their  action  is  sometimes  observed  in 
the  control  tubes  of  agglutination  tests,  especially  if  the  bacteria 
are  grown  in  media  which  have  become  concentrated  by  drying. 
This  effect  is  more  easily  demonstrated  with  certain  salts  of  the 
heavy  metals  than  with  sodium  chloride.  The  following  experi- 
ment will  serve  to  illustrate  the  effect  of  electrolytes  on  the  agglu- 
tination of  sensitized  and  unsensitized  bacteria  : 

Reagents : 

1.  Serum  of  animal  immunized  to  B.  typhosus. 

2.  B.  typhosus  —  24  hour  agar  slant. 

3.  Distilled  water. 


74          LABORATORY  COURSE  IN  SERUM  STUDY 

4.  10  per  cent  salt  solution. 

5.  Copper  sulphate  solution  0.8  per  cent. 

Prepare  typhoid  suspension  (killed  with  formalin  as  in  previous 
lesson  for  the  sake  of  safety) .  Place  in  each  of  two  centrifuge  tubes,  with 
pointed  tip,  2  c.c.  of  the  suspension.  To  tube  A  add  2  c.c.  of  agglutinat- 
ing serum  diluted  1-50.  To  tube  B  add  2  c.c.  of  distilled  water.  Allow 
the  tubes  to  stand  at  37°  C.  for  30  minutes.  Centrifugalize  the  tubes  at 
high  speed  until  the  supernatant  fluid  is  clear.  Resuspend  the  sedi- 
ments in  5  c.c.  of  distilled  water  and  again  centrifugalize.  To  the  washed 
sediments  add  2  c.c.  of  distilled  water  and  draw  the  mixture  repeatedly  in 
and  out  of  the  capillary  pipette  in  order  to  break  up  the  clumps  and 
obtain  an  even  suspension.  Set  up  the  following  tests  in  agglutination 
tubes : 

1.  Sediment  A  0.5  c.c.     Distilled  water  0.5  c.c. 

2.  Sediment  A  0.5  c.c.     10%  salt  sol.  0.09  c.c.     Dist.  water  0.4  c.c. 

Copper  sulphate  sol.  0.8  %  Distilled  water 

3.  Sediment  A  0.5  c.c.  0.02  c.c.  0.5  c.c. 

4.  Sediment  B  0.5  c.c.  0.02  c.c.  0.5  c.c. 

5.  Sediment  B  0.5  c.c.  0.1    c.c.  0.5  c.c. 

6.  Sediment  B  0.5  c.c.  0.5  c.c. 

The  tubes  are  placed  in  the  water  bath  at  37°  for  one  hour  and  then 
observed.  Tubes  2,  3  and  5  should  show  agglutination. 

This  experiment  shows  that  electrolytes  are  necessary  for  the 
appearance  of  agglutination  and  that  bacteria  even  though  they 
may  have  united  with  the  specific  serum  agglutinins  do  not  clump 
in  the  absence  of  such  salts.  It  is  the  mainstay  of  Bordet's  view 
of  agglutination  as  a  "two-phase"  reaction  in  which  one  step  is 
the  union  with  agglutinins,  the  other  the  actual  clumping  brought 
about  by  the  salts. 

Each  student  needs:  8  watch  glasses,  2  centrifuge  tubes,  6  f-inch  test 
tubes,  glass  tubing,  test  tube  rack. 

Bacillus  typhosus,  24-hr  broth  culture,  24-hr,  agar  slant. 

LESSON  XI 
ABSORPTION  OF  BACTERIAL  AGGLUTININS 

THE  serum  of  an  animal  immunized  simultaneously  with  two 
species  of  bacteria  develops  agglutinative  power  for  each  of  these 
species.  The  agglutinins  for  the  two  species  may  be  shown  to 
be  distinct  by  treating  the  serum  with  a  heavy  emulsion  of  one  of 


76  LABORATORY   COURSE    IN    SERUM   STUDY 

them,  and  subsequently  removing  the  agglutinated  bacteria  by 
centrifugation.  The  agglutinins  for  this  species  having  been  ab- 
sorbed by  the  bacteria  will  be  removed  by  this  procedure,  leaving 
the  agglutinins  for  the  species  not  used  in  the  absorption  unin- 
fluenced. For  the  complete  removal  of  agglutinins  it  is  necessary 
to  leave  the  serum  in  contact  with  the  bacteria  for  a  long  period 
and  often  to  repeat  the  treatment  of  the  serum  with  bacterial 
suspension  several  times.  These  facts  are  illustrated  in  Experi- 
ment 1. 

The  experiment  illustrates  the  general  principle  that  an  animal 
may  produce  specific  and  distinct  antibodies  simultaneously 
against  a  number  of  different  antigens. 

Experiment  1 

ABSORPTION  OF  AGGLUTININS  FROM  THE  SERUM  OF  AN  ANIMAL  IM- 
MUNIZED AGAINST  Two  SPECIES  OF  BACTERIA 

Reagents : 

1.  Serum  of  an  animal  simultaneously  immunized  against  Bacillus 
typhosus  and  Bacillus  coli.     The  students  who  have  immunized  this 
animal  should  bleed  it  from  the   carotid   at   a  preceding  lesson   and 
should  distribute  the  serum  to  other  members  of  the  class,  diluted  1-10 
with  salt  solution.     Each  student  requires  5  c.c.  of  this  dilution. 

2.  Thick  suspension  of  typhoid  bacilli  killed  by  heating  one  hour  at 
55°  C. 

3.  Thick  suspension  of  colon  bacilli  killed  by  heating  one  hour  at 
60°  C. 

Both  these  suspensions  should  be  used  undiluted  for  the  absorption, 
and  in  a  dilution  of  1-10  for  titrating  the  agglutination. 

4.  Salt  solution. 

A.     PRELIMINARY  TITRATION  OF  AGGLUTINATIVE  POWER  OF  IMMUNE 

SERUM 

The  dilutions  of  serum  required  for  this  test  should  be  prepared 
in  f-inch  test  tubes  according  to  a  protocol  to  be  made  out  by  the  indi- 
vidual student.  Before  making  any  such  series  of  dilutions  it  is  highly 
important  to  write  out  plainly  the  amount  of  serum  and  salt  solution 
to  be  placed  in  each  tube  in  order  to  avoid  errors. 


78  LABORATORY   COURSE   IN    SERUM   STUDY 

Set  up  the  following  experiment  in  agglutination  tubes : 

Tube  No.  1  Serum  (1-20)  0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

2  Serum  (1-100)  0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

3  Serum  (1-500)  0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

4  Serum  (1-2000)  0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

5  Salt  solution  0.5  c.c.  -f-  Typhoid  suspension  0.5  c.c. 

6  Serum  (1-20)  0.5  c.c.  +  Colon  suspension       0.5  c.c. 

7  Serum  (1-100)  0.5  c.c.  +  Colon  suspension       0.5  c.c. 

8  Serum  (1-500)  0.5  c.c.  +  Colon  suspension       0.5  c.c. 

9  Serum  (1-2000)  0.5  c.c.  +  Colon  suspension       0.5  c.c. 

10  Salt  solution       0.5  c.c.  -f  Colon  suspension       0.5  c.c. 

Incubate  one  hour  at  37°  C.  and  record  the  results. 

B.  ABSORPTION 
Place  in  small  centrifuge  tubes : 

A.  Serum  (1-10)  1.5  c.c.  +  Thick  typhoid  suspension  1.5  c.c. 

B.  Serum  (1-10)  1.5  c.c.  +  Thick  colon  suspension      1.5  c.c. 

Incubate  one  hour.  (For  complete  absorption,  incubation  in  the 
thermostat  should  be  followed  by  12  or  more  hours  in  the  refrigerator.) 
Centrifugalize  at  high  speed  and  remove  the  clear  supernatant  fluid.1 

C.    TITRATION   OF  THE   SERUM  AFTER  ABSORPTION 

The  supernatant  fluid  obtained  by  centrifugation  should  be  tested 
undiluted  (it  corresponds  to  a  1-20  dilution  of  the  original  serum)  against 
the  same  organism  used  for  absorption,  and  as  the  absorption  is  often 
incomplete  after  this  short  incubation  a  similar  test  should  be  set  up 
of  the  supernatant  fluid  diluted  1-5  (corresponding  to  a  1-100  dilution 
of  the  original  serum).  The  supernatant  fluid  should  also  be  tested 
against  the  organism  not  used  in  absorption  in  the  highest  dilution  in 
which  agglutination  was  found  in  the  preliminary  tit  ration. 

1.  Supernatant  fluid  A  0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

2.  Supernatant  fluid  A  (1-5)  0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

3.  Supernatant  fluid  A  0.5  c.c.  +  Colon  suspension      0.5  c.c. 

4.  Supernatant  fluid  in  high- 

est dilution  in  which  it 
agglutinated  colon  in  pre- 
liminary titration  0.5  c.c.  +  Colon  suspension  0.5  c.c. 

1  It  should  be  noted  that  absolutely  complete  removal  of  agglutinins  is  not 
possible  except  by  many  times  repeated  absorption. 


80  LABORATORY  COURSE  IN  SERUM  STUDY 

5.  Supernatant  fluid  B  0.5  c.c.  +  Colon  suspension       0.5  c.c. 

6.  Supernatant  fluid  B  (1-5)  0.5  c.c.  -f  Colon  suspension       0.5  c.c. 

7.  Supernatant  fluid  B  0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

8.  Supernatant  fluid  in  high- 

est dilution  in  which  it 
agglutinated  typhoid  in 
original  titration  0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

9.  Salt  solution  .    0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 
10.   Salt  solution  0.5  c.c.  +  Colon  suspension      0.5  c.c. 

Incubate  one  hour  at  37°  C.  and  record  results. 

Tubes  1,  2,  5  and  6  should  show  no  agglutination  if  absorption  has 
been  complete.  If  all  the  agglutinin  has  not  been  absorbed,  however, 
there  may  be  clumping  in  tubes  1  and  5.  The  bacteria  in  tubes  3,  4,  7 
and  8  should  be  agglutinated. 

GROUP  AGGLUTININS 

The  serum  of  animals  highly  immunized  to  one  species  of 
bacteria  will  often  agglutinate  closely  related  bacteria,  though 
this  action  is  invariably  weaker  than  the  agglutinative  power  for 
the  bacteria  used  in  immunizing  the  animal. 

Thus  the  serum  of  an  animal  which  before  treatment  agglu- 
tinated Bacillus  coli  in  dilutions  of  1-20  and  Bacillus  typhosus  in 
dilutions  of  1-5,  after  immunization  with  Bacillus  typhosus  may 
agglutinate  Bacillus  coli  as  highly  as  1-100,  and  Bacillus  typhosus 
up  to  1-10,000  or  more.  This  indicates  a  close  similarity  in  the 
chemical  constituents  of  the  two  antigens  —  the  Bacillus  typhosus 
antigen  containing  small  amounts  of  the  protein  present  in  Bacillus 
coli.  Absorption  with  Bacillus  typhosus  will  remove  both  the 
Bacillus  typhosus  and  the  colon  agglutinins.  Absorption  with 
Bacillus  coli  will  take  out  only  the  colon  agglutinin,  the  serum  still 
agglutinating  Bacillus  typhosus  1-10,000. 

The  serum  here  contains  at  least  two  types  of  agglutinins,  the 
so-called  major  or  specific  agglutinin  which  reacts  only  with  the 
homologous  bacteria,  and  the  minor  or  group  agglutinins  which 
react  both  with  the  homologous  bacteria  and  with  the  allied 
species.  If  such  a  serum  is  added  to  a  suspension  of  the  homolo- 
gous bacteria  both  major  and  minor  agglutinins  are  absorbed 


82          LABORATORY  COURSE  IN  SERUM  STUDY 

and  the  supernatant  fluid  obtained  after  centrifugation  has  lost 
its  agglutinative  power  both  for  the  homologous  strains  and  for 
allied  species.  If,  on  the  other  hand,  the  serum  is  treated  with  a 
suspension  of  the  one  allied  species,  the  minor  agglutinins  only 
are  absorbed.  The  supernatant  fluid  then  shows  undiminished 
agglutinative  power  for  the  homologous  strain,  but  does  not  ag- 
glutinate the  allied  strain  used  in  absorption.  These  facts  are 
brought  out  in  Experiment  2. 

Experiment  2 

ABSORPTION  OF  MAJOR  AND  MINOR  AGGLUTININS  FROM  SERUM  OF 
AN  ANIMAL  IMMUNIZED  AGAINST  A  SINGLE  SPECIES 

Reagents : 

1.  Serum  of  an   animal   immunized   to   Bacillus   typhosus.     The 
serum  must  be  known  to  contain  group  agglutinins  for  Bacillus  coli.1 

2.  Two  specimens  of  the  same  serum  in  1-20  dilution  which  have 
stood  in  contact  over  night  with  typhoid  and  with  colon  bacilli  re- 
spectively.    These  may  be  prepared  on  the  previous  day  by  the  in- 
structor.    Heavy  suspensions  of  typhoid  and  colon  bacilli  are  first 
prepared  by  washing  off  the  growth  of  a  number  of  agar  bottles  and 
heating  for  one  hour  at  56°  and  60°  respectively.     To  each  of  these  sus- 
pensions is  added  an  equal  volume  of  a  1-10  dilution  of  agglutinating 
serum.     The  tubes  are  incubated  for  one  hour  at  37°,  shaken  thoroughly 
and  placed  in  the  ice  box  over  night.     They  should  be  shaken  once  or 
twice  during  the  period  of  24  hours.     They  are  then  centrifugalized  and 
the  supernatant  fluids  removed  and   used  in  the  second  part  of  the 
experiment. 

3.  Suspension  of  killed  typhoid  bacilli  as  in  Experiment  1. 

4.  Suspension  of  killed  colon  bacilli  as  in  Experiment  1. 

5.  Salt  solution. 

(1)  Titrate  the  original  antityphoid  serum  against  both  typhoid 
and  colon  emulsions  according  to  the  following  protocol.  This  gives  a 
basis  for  comparison  with  a  similar  test  on  the  same  serum  after  it  has 
been  in  contact  with  the  bacteria. 

1  To  insure  successful  class  work  this  must  be  previously  ascertained  in 
each  case,  since  individual  animals  may  vary  considerably  in  regard  to  the  pro- 
duction of  minor  agglutinins. 


84  LABORATORY   COURSE    IN    SERUM   STUDY 

1.  Serum  (1-10)      0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

2.  Serum  (1-10)      0.5  c.c.  +  Colon  suspension       0.5  c.c. 

3.  Serum  (1-50)      0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

4.  Serum  (1-50)      0.5  c.c.  -f  Colon  suspension      0.5  c.c. 

5.  Serum  (1-200)    0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

6.  Serum  (1-200)    0.5  c.c.  +  Colon  suspension      0.5  c.c. 

7.  Serum  (1-1000)  0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

8.  Serum  (1-4000)  0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

9.  Salt  solution       0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 
10.  Salt  solution       0.5  c.c.  +  Colon  suspension      0.5  c.c. 

Incubate  one  hour  at  37°  C.  and  observe. 

(2)  Set  up  the  following  tests  with  the  same  serum,  from  which 
in  this  case,  however,  the  agglutinins  have  been  removed  by  ab- 
sorption : 

1.  Serum  absorbed  with  typhoid  0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

2.  Serum  absorbed  with  typhoid  0.5  c.c.  +  Colon  suspension      0.5  c.c. 

3.  Serum  absorbed  with  colon      0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

4.  Serum  absorbed  with  colon      0.5  c.c.  +  Colon  suspension      0.5  c.c. 

5.  Serum  absorbed  with  colon 

in  the  highest  dilution 
which  agglutinated 
typhoid  bacilli  before 
absorption  0.5  c.c.  +  Typhoid  suspension  0.5  c.c. 

Incubate  for  one  hour  and  observe. 
Each  student  needs: 

25  J-inch  test  tubes,  8  1-c.c.  pipettes,  test  tube  rack,  35  £-inch 
test  tubes,  4  centrifuge  tubes,  test  tube  racks. 

LESSON  XII 

EFFECT    OF    HJEMOLYTIC    AND    H^EM  AGGLUTINATIVE 

SERA  IN  VIVO 

1.     PRELIMINARY  TESTS 

A  RABBIT  receives  at  four-day  intervals  four  intravenous  injections 
of  2  c.c.  of  washed,  cat's  red  blood  cells.  For  obtaining  the  cells  a  cat 
under  ether  is  bled  from  the  carotid  or  jugular.  The  defibrinated  cat's 
blood  if  sterile  can  be  kept  four  days  and  thus  used  for  two  injections. 
Seven  to  ten  days  after  the  last  injection  the  rabbit  is  bird  and  the 
hsemolytic  and  agglutinative  power  of  the  inactivated  immune  serum  for 
cat's  cells  is  tested. 


86 


LABORATORY  COURSE  IN  SERUM  STUDY 


The  following  test  is  made  to  determine  the  haemolytic  power  of  the 
anti-cat  rabbit  serum : 


TUBE 

IMMUNE  SERUM 

GUINEA  PIG  SERUM 
OR  CAT'S  SERUM, 
FRESH  DILUTED 
1-10 

SALINE  UP  TO 

5%  WASHED  CAT'S 
RED  BLOOD  CELLS 

1 

Diluted  1-10    0.5 

0.5  c.c. 

2  c.c. 

0.5  C.C. 

2 

Diluted  1-10    0.1 

0.5  c.c. 

2  c.c. 

0.5  c.c. 

3 

Diluted  1-100  0.5 

0.5  c.c. 

2  c.c. 

0.5  c.c. 

4 

Diluted  1-100  0.1 

0.5  c.c. 

2  c.c. 

0.5  c.c. 

5 

Diluted  1-400  0.2 

0.5  c.c. 

2  c.c. 

0.5  c.c. 

6 

Diluted  1-400  0.1 

0.5  c.c. 

2  c.c. 

0.5  c.c. 

In  testing  the  agglutinative  power  set  up  a  similar  series  but  without 
fresh  serum. 

II.   IN  Vivo  EFFECT  OF  H^MOLYTIC  SERUM 

Etherize  two  cats  and  inject  1  c.c.  of  immune  serum  into  one  and 
3  c.c.  into  the  other.  Give  the  injections  directly  into  the  saphenous 
vein  with  a  fine  hypodermic  needle.  Put  the  first  cat  into  a  clean  cage 
with  a  dram  for  the  collection  of  urine  after  6,  24  and  48  hours.  (The 
first  urine  will  con  tain  large  amounts  of  haemoglobin,  the  last  bile.)  Half 
an  hour  after  the  injection  collect  a  specimen  of  the  cat's  serum  by  prick- 
ing the  ear  vein.  Centrifugalize  and  observe  the  blood-tinged  serum. 
Make  smears  of  the  blood,  stain,  and  note  with  oil  immersion  lens  the 
phagocyted  red  cells  in  some  of  the  leucocytes.  The  second  cat  will 
probably  die  at  once.  If  so,  autopsy  immediately  and  look  for  clumps 
of  agglutinated  red  cells  in  portal  and  pulmonary  veins. 

To  demonstrate  the  in  vivo  effect  of  complement,  give  a  rabbit  an 
intravenous  injection  of  2.0  c.c.  of  25  %  suspension  of  sensitized  sheep 
blood  cells.  (In  sensitizing  the  cells,  regard  0.1  c.c.  of  25  %  cells  as  a 
unit  and  add  two  units  of  amboceptor  for  every  unit  of  cells.  After 
the  cells  have  stood  in  contact  with  the  amboceptor  for  one  half  hour, 
centrifugalize  them  and  make  them  up  to  the  original  volume  of  25  % 
suspension). 

After  a  half  hour  collect  a  specimen  of  serum  from  the  rabbits' 
ear  and  examine  for  dissolved  haemoglobin.  Make  a  smear  of  the  blood 
and  examine  for  phagocytosis.  Examine  the  rabbit's  urine  after  6, 
24  and  48  hours. 

The  experiments  in  this  lesson  need  not  be  done  by  the  entire  class 
but  can  be  done  as  a  demonstration  by  a  small  group  of  students  working 
with  the  instructor. 


88  LABORATORY    COURSE    IN    SERUM    STUDY 

Materials: 

5  Cats 

2  Rabbits 

Hypodermic  syringe 

Fresh  guinea  pig  serum 

Test  tubes 
4  f-inch  test  tubes 

6  £-inch  test  tubes 
Test  tube  racks 

Cover  slips,  microscope  slides 
Blood  stain  (Wright's,  Giemsa's  or  Jenner's) 
1  Centrifuge  tube 

LESSON  XIII 

ISOKEMOLYSIS  AND  ISOAGGLUTINATION 

TRANSFUSION  TESTS 

ANTIBODIES  in  the  blood  of  one  animal  capable  of  acting  on 
the  blood  of  another  animal  of  the  same  species  are  called  iso- 
antibodies.  Isoagglutinins  and  isohsemolysins  sometimes  occur 
naturally  and  they  can  often  be  made  to  appear  by  immunization 
of  animals  with  the  blood  of  other  animals  of  the  same  species. 
They  appear  with  a  peculiar  regularity  which  indicates  that  there 
are  certain  groups  of  individuals  within  one  and  the  same  species 
whose  blood  is  strictly  homologous  only  with  other  members  of  the 
same  group.  Among  human  beings  there  are  four  such  groups, 
sharply  defined,  permanent  and  hereditary.  The  existence  of 
these  antibodies  is  of  practical  importance  in  connection  with 
blood  transfusion  because  more  or  less  serious  results  may  ensue 
if  a  patient  is  transfused  with  the  blood  of  a  donor  who  belongs  to 
another  group  and  whose  blood  is  therefore  either  haemolytic 
or  hsemagglutinating  for  the  patient,  or  vice  versa.  (See  table 
in  "Infection  and  Resistance,"  page- 238.) 

Before  transfusion,  therefore,  unless  the  urgency  of  the  case  for- 
bids, a  number  of  prospective  donors  are  tested,  and  one  is  chosen 
whose  blood  does  not  lake  or  agglutinate  when  mixed  with  that  of 
the  patient.  The  serum  of  each  donor  must  be  tested  with  the 
blood  cells  of  the  patient  and  the  serum  of  the  patient  with  the 
cells  of  each  donor. 

There  are  two  methods  of  collecting  blood  for  these  tests : 


90       LABORATORY  COURSE  IN  SERUM  STUDY 

(A)  When  possible  from  a  vein,  3  to  5  c.c.  in  a  test  tube  for  serum, 
10  drops  in  5  c.c.  of  a  solution  containing  1  per  cent  sodium 
citrate  and  0.9  per  cent  sodium  chloride,  for  the  red  cell  emulsion. 

(B)  By  pricking  the  finger  with  a  Hagedorn  needle  and  milking 
out  from  1  to  2  c.c.  of  blood  which  is  collected  in  a  Wright  capsule 
for  serum  and  10  drops  in  citrate-salt  solution  for  cells. 

Students  make  Wright  capsules  and  collect  specimens  of  blood 
from  each  other.  The  capsules  are  of  heavy  glass  tubing,  7  to  8  mm. 
in  diameter,  about  10  cm.  long,  and  drawn  to  a  capillary  opening  of 
about  1  mm.  diameter  at  each  end.  They  are  laid  on  the  table  and  drops 
of  blood  expressed  from  the  finger  are  allowed  to  enter  them  by  capillary 
action.  When  the  capsule  is  two  thirds  full,  the  blood  is  allowed  to  clot 
and  the  open  end  of  the  capsule  is  sealed  in  the  flame,  great  care  being 
taken  not  to  heat  the  blood.  Serum  is  obtained  by  centrifugalizing  the 
capsules  and  cutting  them  with  a  small  file. 

The  cell  emulsions  are  prepared  by  washing  the  citrated  blood  twice 
with  salt  solution  and  making  up  to  5  per  cent  by  comparing  the  opacity 
with  that  of  a  5  per  cent  sheep  cell  suspension,  or  by  measuring  with 
capillary  pipette  1  volume  of  sedimented  human  cells  and  19  volumes 
of  saline.1 

Each  student  should  make  12  Wright  capillary  pipettes  of  strong 
glass  tubing,  4  to  5  mm.  in  diameter.  These  pipettes  are  made  by  cut- 
ting the  tubing  into  pieces  about  10  cm.  inches  long,  heating  the  center  of 
each  piece  to  melting  and  drawing  out  to  a  capillary  of  about  1  mm. 
diameter  and  10-12  cm.  inches  long.  The  capillary  is  then  broken  at  the 
middle,  giving  two  pipettes.  Adjust  nipple  to  the  pipettes.  Make  a 
mark  on  the  capillary  end  of  each  pipette  near  the  hilt.  Draw  up  1 
volume  of  serum  to  the  mark,  then  a  small  buble  of  air,  then  another 
volume  of  serum,  and  another  bubble  of  air,  and  then  a  volume  of  the 
cells  which  are  to  be  mixed  with  the  serum ;  (the  serum  is  used  in  excess 
because  in  this  way  it  is  sometimes  possible  to  detect  a  weak  hsemolytic 
action  which  would  otherwise  be  overlooked  on  account  of  the  low  com- 
plement activity  of  human  blood).  Draw  the  mixture  into  the  body  of 
the  pipette  and  seal  the  capillary  tip  in  the  flame.  Mix  the  fluid  by 
rotating  the  pipette.  Remove  the  nipple.  Throughout  these  steps 
keep  the  pipette  approximately  horizontal  so  that  the  fluid  remains  in 
the  body  of  it  and  does  not  flow  to  either  end.  Seal  the  open  end  of  the 
pipette  by  dipping  in  melted  paraffine.  Mark  the  pipette  with  a  glass 
pencil,  using  the  number  assigned  to  the  serum  as  the  numerator,  that 
assigned  to  the  ce.lls  as  the  denominator,  of  a  fraction. 

1  It  is  not  necessary  in  practical  work  to  wash  the  cells.  Three  or  four  drops  of 
blood  in  citrate  salt  solution  furnishes  satisfactory  material. 


92 


LABORATORY  COURSE  IN  SERUM  STUDY 


Each  student  tests  one  specimen  of  serum  against  10  different  indi- 
viduals' red  cells.  Make  a  control  of  each  serum  with  its  own  red  cells. 
Lay  all  the  pipettes  on  their  sides  on  a  tray  and  incubate  at  37.5°  C. 
for  one  hour.  Then  make  observations  for  haemolysis  or  agglutination 
and  make  the  results  of  all  the  tests  into  a  table  to  illustrate  Land- 
steiner's  law  of  the  groupings  of  human  isoagglutination. 

By  the  original  classification  of  blood  groups  the  groupings  may  be 
arranged  as  follows  on  the  assumption  that  there  are  two  distinct  ag- 
glutinins  and  corresponding  agglutinogens: 


SERA                                         I 
•  AGGLUTININS                            aft 

II           III           VI 
a            &            o 

.Cells 
I 
II 
III 
IV 

Agglutinogens 
O 
B 
A 
AB 

+           +           +           - 

From  this  table  the  student  can  determine  the  grouping  of  his  tests. 
It  will  be  noted  that  any  individual  can  be  grouped  by  testing  his  cells 
against  sera  of  groups  II  and  III.  Study  this  out  on  the  table  above. 
Every  serologist  should  know  his  own  group. 

The  students  should  carry  out  a  few  simplified  tests  by  mixing  a 
drop  of  blood  suspension  and  a  drop  of  serum  on  a  slide.  The  slide 
method  is  sufficient  for  all  practical  purposes. 

The  class  needs:  « 

Heavy  glass  tubing  7-8  mm.  diameter. 
Heavy  glass  tubing  4-5  mm.  diameter. 

Litre  1  per  cent  sodium  citrate  in  0.6  per  cent  sodium  chloride. 
Litre  0.9  per  cent  sodium  chloride  solution. 
Triangular  files. 

Rubber  nipples  to  fit  5  mm.  tubing  (one  nipple  for  each  student). 
Hard  paraffine  in  casserole. 


94 


LABORATORY  COURSE  IN  SERUM  STUDY 


LESSON  XIV 
TITRATION  OF  PRECIPITATING  SERA  FROM  RABBITS 

SPECIFICITY  OF  PRECIPITINS 

PRECIPITATING  sera  are  active  against  proteins  in  high  dilu- 
tions. Their  specificity  is  great  but  is  limited  by  group  reactions 
so  that  a  precipitating  serum  for  the  protein  of  any  species  of 
animal  or  bacteria  will  give  precipitation,  with  closely  related 
species  in  low  but  not  in  high  dilutions.  On  this  is  based  Nuttall's 
well-known  study  of  blood  relationships. 

Students  working  in  groups  bleed  serum-treated  rabbits  (Lesson  I) 
from  the  carotid  10  days  after  the  last  injection,  taking  about  20  c.c. 
of  blood  in  small  centrifuge  tubes.  The  animal  should  be  in  a  fasting 
state  in  order  that  the  serum  will  be  clear.  Allow  the  blood  to  clot  and 
centrifugalize. 

Experiment  1 

One  half  of  the  students  in  the  class  make  dilutions  in  large  test 
tubes  of  sheep  serum  or  human  serum  respectively  (according  to  par- 
ticular serum  with  which  rabbit  has  been  treated)  as  follows:  1-10,  1-20, 
1-50,  1-100,  1-200,  1-500,  1-1000.  In  order  to  test  the  specificity  of  the 
reaction  the  other  half  of  the  students  make  similar  dilutions  of  serum  from 
other  species  of  animal  —  guinea  pig,  dog,  cat,  sheep,  horse,  as  available. 

Then  set  up  the  following  mixtures  in  small  "precipitation"  tubes 
(tubes  of  about  5-8  mm.  diameter) : 


TUBE 

IMMUNE  RABBIT  SERUM 

ANTIOEN  (PRECIPITINOOEN) 

1 

0.1  c.c. 

Undiluted              0.1  c.c. 

2 

0.1  c.c. 

1-20                    0.1  c.c. 

3 

0.1  c.c. 

1-50                   0.1  c.c. 

4 

0.1  c.c. 

1-100                 0.1  c.c. 

5 

0.1  c.c. 

1-200                 0.1  c.c. 

6 

0.1  c.c. 

1-500                 0.1  c.c. 

7 

0.1  c.c. 

1-1000                0.1  c.c. 

8 

0.1  c.c. 

Salt  solution           0.1  c.c. 

9 

Salt  solution  0.25  c.c. 

Undiluted  antigen  0.1  c.c. 

In  making  these  mixtures,  put  the  anti-serum  into  the  tubes  first 
and  then  run  the  dilution  into  the  tubes  with  a  nipple  pipette  in  such  a 
way  that  the  latter  is  layered  over  the  former  as  in  a  Heller's  test  for 
albumin  in  urine.  This  is  the  so-called  "ring  test."  .  The  precipitum 
shows  as  a  white  line  at  the  contact  of  the  two  fluids.  After  this  ring 


96  LABORATORY    COURSE    IN    SERUM    STUDY 

has  formed,  the  tubes  can  be  shaken  up  and  the  ordinary  precipitin  reac- 
tion (i.e.,  turbidity  and  flocculation)  can  be  observed.  Note  the  results 
after  one  half  hour  and  one  hour  at  37°  C.  and  again  after  12  hours  in 
the  ice  box.  Is  there  a  "pro  zone"?  • 

Experiment  2 

EFFECT  OF  HEAT  ON  PRECIPITINS 

Heat  slowly  inactivates  precipitins.  They  cannot  be  reactivated 
by  fresh  serum  (alexin, complement), in  this  regard  differing  from  haemoly- 
sins  and  bactericidal  antibodies. 

Heat  0.5  c.c.  of  precipitin  serum  for  two  hours  at  60°  C.  Mix 
0.1  c.c.  of  the  heated  serum  with  0.1  c.c.  of  that  dilution  of  the  antigen 
which  was  found  in  Experiment  1  to  give  the  optimum  precipitation. 

Mix  0.1  c.c.  of  the  heated  serum  with  0.1  c.c.  of  fresh  normal  rabbit 
serum  and  test  this  mixture  also  with  the  optimum  dilution  of  antigen. 
Make  observations. after  1  hour  at  37°  C.  and  again  after  12  hours  in  ice 
box. 

For  each  student: 

2  c.c.   Immune  serum   (rabbit  whose  preparation  was  begun  in 

Lesson  I). 
1  c.c.  Antigen  serum  (human,  sheep  or  horse  serum  for  half  the 

class,  other  animals'  sera  for  the  other  half). 
Salt  solution. 

12  Precipitation  tubes.    Test  tube  rack.    Nipple  pipette. 
6  one-c.c.  graduated  pipettes. 
0.1  c.c.  normal  rabbit  serum,  fresh, 
8  f-inch  test  tubes. 

Precipitins  can  also  be  produced  against  proteins  heated  to 
70°  or  over.  For  the  principles  of  such  experiments  see  Infection 
and  Resistance,  page  260. 


LESSON  XV 
FORENSIC  PRECIPITIN  TEST 

THE  precipitin  reaction  is  used  in  medicolegal  work  to  identify 
blood  stains  and  to  detect  adulterations  or  substitutions  in 
meat  or  other  food  products.  Since  the  specificity  of  the  precipi- 
tin reaction  is  subject  to  limitations  similar  (though  not  as 
marked)  to  those  discussed  in  the  case  of  agglutination,  the  pre- 
cipitating serum  used  in  forensic  tests  must  be  very  potent. 
Preliminary  test  should  show  that  the  serum  gives  distinct  cloud- 
ing with  its  homologous  antigen  diluted  1  to  1000,  within  five  or 
ten  minutes  at  room  temperature. 

An  effort  is  made  to  get  the  suspected  protein  into  approxi- 
mately this  dilution. 

1 

Each  student  gets : 

2.0  c.c.  antihuman  serum. 

2.0  c.c.  antisheep  serum. 

0.4  c.c.  normal  rabbit  serum. 

Two  specimens  of  material  with  dry  blood  to  be  identified.  (To  some 
of  the  students  sheep  blood  is  given ;  to  others  human  blood  or  that  of 
other  species.) 

From  each  of  the  specimens  upon  which  blood  has  been  dried  the 
blood  must  be  soaked  off  in  salt  solution.  This  can  be  done  by  placing 
the  material  in  a  test  tube  with  5  c.c.  salt  solution  and  shaking  for 
10  or  15  minutes  until  the  solution  presents  a  lasting  foam.  This  is 
evidence  that  the  protein  is  in  solution  in  a  concentration  of  at  least  1  part 
in  1000.  Old  specimens  must  often  be  left  in  salt  solution  in  the  refrig- 
erator for  24  hours  or  longer.  Such  prolonged  soaking  is  done  in  the 
refrigerator  in  order  to  avoid  destruction  of  the  antigen  by  bacterial 
growth.  Since  the  nature  of  the  reaction  makes  it  necessary  that  the 
material  to  be  examined  shall  be  absolutely  clear,  it  is  now  cleared  by 
filtration  or  centrifugation ;  if  there  is  enough  material  the  acetic-acid- 
and-boiling  test  can  be  done  and  the  cloud  compared  in  heaviness  with 
a  1-1000  dilution  of  serum  of  the  suspected  variety  of  blood,  similarly 


100          LABORATORY  COURSE  IN  SERUM  STUDY 

treated.  This  gives  a  rough  estimate  of  the  concentration  of  the  antigen. 
If  the  solution  seems  much  too  concentrated,  dilute  it  until  it  approaches 
1-1000,  —  i.e.  the  limit  of  its  still  presenting  a  lasting  foam  on  shaking. 
Place  the  antiserum  in  the  tube  first  and  make  a  ring  test.  To  do 
this  run  antigen  very  carefully  into  tube  with  a  nipple  pipette  so  that 
the  two  fluids  may  not  be  mixed.  Watch  for  a  white  ring  at  the  line  of 
junction.  Be  careful  to  compare  this  with  the  control,  since  a  slight 
opalescence  is  often  seen  at  the  line  of  junction  of  salt  solution  and 
serum  not  due  to  precipitation. 

2 

1.  Unknown  solution  (a)  0.5  c.c.  +  antihuman  serum    0.2  c.c. 

2.  Unknown  solution  (a)  0.5  c.c.  +  normal  rabbit 

serum    0.2  c.c. 

3.  Unknown  solution  (6)  0.5  c.c.  +  antihuman  serum   0.2  c.c. 

4.  Unknown  solution  (6)  0.5  c.c.  -f  normal  rabbit 

serum    0.2  c.c. 

5.  Known  human  serum  (1-1000)  0.5  c.c.  -f  antihuman  serum    0.2  c.c. 

6.  Salt  solution  0.5  c.c.  +  antihuman  serum    0.2  c.c. 
Observe  the  tubes  for  15  or  20  minutes  at  room  temperature.     Then 

incubate  and  observe.     After  30  minutes  shake  up  and  observe  for 
cloudiness  and  flocculation. 

3 

Set  up  a  similar  series  with  antisheep  instead  of  antihuman  serum. 
Let  each  student  report  whether  either  of  the  specimens  he  received  is 
sheep  or  human. 

In  order  that  conclusions  may  be  definitely  drawn  from  such  a  test 
it  is  necessary  that  the  tubes  containing  the  unknown  solution  (say  it  is 
suspected  of  being  human  blood)  and  the  known  human  blood,  1-1000 
control,  should  show  a  definite  reaction  within  not  longer  than  10  min- 
utes at  room  temperature.  Antisera  too  weak  to  show  such  a  sharp 
reaction  with  controls  of  the  known  protein  are  not  suitable  for  such 
tests. 

Each  student  needs: 

6  f-inch  test  tubes.     12  Precipitation  tubes  6  mm.  in  diameter. 
Capillary  pipettes. 

LESSON  XVI 

BORDET-GENGOU  PHENOMENON— COMPLEMENT 

FIXATION 

THE  Bordet-Gengou  phenomenon  is  the  prototype  of  all  com- 
plement fixation  methods.  It  depends  on  the  fact  that  antigens 


102 


LABORATORY    COURSE    IN    SERUM    STUDY 


(bacterial  and  others)  in  the  presence  of  their  specific  antisera 
will  fix  complement  so  that  the  subsequently  added  red  cells 
sensitized  by  their  appropriate  hsemolytic  immune-body  will  fail 
to  be  laked,  because  no  free  complement  is  available.  Neither  the 
antigen  alone  nor  the  antibody  alone  will  fix  complement  in  this 
manner. 

Unfortunately  bacterial  cell  bodies  (like  many  other  sub- 
stances) if  in  sufficiently  large  amounts  will  of  their  own  accord, 
probably  for  purely  physical  reasons,  absorb  complement,  in  a 
non-specific  way,  i.e.,  without  the  presence  of  a  specific  antibody. 

The  immune  sera  likewise  have  often  the  power  of  absorbing 
complement  in  a  non-specific  way  in  the  absence  of  antigen  and 
this  power  must  be  determined  by  preliminary  titration  in  order 
that  allowance  may  be  made  for  it  in  the  main  test. 

For  this  reason  before  doing  a  Bordet-Gengou  reaction  (and, 
as  will  be  seen  later,  in  all  complement  fixation  work)  it  is  neces- 
sary to  determine  the  degree  to  which  such  non-specific  comple- 
ment fixation  will  take  place  with  the  given  antigen  and  to  use 
in  the  actual  test  an  amount  too  small  to  give  such  a  false  reaction. 


Experiment  1 

PRELIMINARY  DETERMINATION  OF  THE  NON-SPECIFIC  BINDING  POWER 
OF  THE  BACTERIAL  ANTIGEN 

Emulsify  a  24  hour  slant  of  typhoid  bacilli  in  10  c.c.  of  saline  solu- 
tion.   Heat  at  56°  C  for  30  minutes. 


TUBE 

BACTERIAL  EMULSION 

COMPLEMENT 

1 
2 
3 

4 
5 
6 

0.4  C.C. 
0.2 
0.1 
1/10  dilution  of  above 
0.8 
0.5 
0.2 

2u 

nits1 

1  Determined  by  preliminary  titration 

Add  sufficient  saline  solution  to  each  tube  so  that  the  final  volume 
after  adding  red  cells  and  amboceptor  shall  equal  2.5  c.c.  Incubate  one 
hour  in  water  bath.  Add  0.5  c.c.  of  5%  reel  cell  suspension  plus  two 
units  of  amboceptor  to  each  tube.  Incubate  for  one  half  hour  in  water 
bath. 


104 


LABORATORY    COURSE    IN    SERUM    STUDY 


Experiment  2 

PRELIMINARY  DETERMINATION  OF  THE  NON-SPECIFIC  BINDING  POWER 
OF  THE  IMMUNE  SERUM 


TUBE 

1/0  DILUTION  OF  SERUM* 

COMPLEMENT 

1 

0.5  c.c. 

2  units 

2 

0.3 

« 

3 

0.2 

« 

4 

0.1 

it 

1  Serum  from  rabbit  immunized  to  typhoid  bacilli.     This  serum  should  be  heated 
to  55°  for  30  minutes  on  day  of  using. 

Follow  same  procedure  as  directed  under  Experiment  1. 


Experiment  3 

Use  that  amount  of  typhoid  suspension  which  is  next  below  the 
amount  which  was  found  to  not  interfere  at  all  with  the  hsemolytic  action 
of  the  complement.  Also  take  that  amount  of  serum  which  lies  next 
below  the  amount  which  did  not  interfere  with  haemolysis.  Let  us  say 
there  was  full  haemolysis  in  Tube  3  of  the  first  set  and  2  of  the  second; 
we  set  up  the  third  as  follows : 


TUBE 

BACTERIAL  SUSPENSION 
1/10  DILUTION 

SERUM 
1/10  DILUTION 

COMPLEMENT 

1 
2 
3 

0.8  C.C. 
0.8 

0.2  c.c. 
0.2  c.c. 

2  units 
2     " 
2     " 

Add  sufficient  saline  solution  to  bring  the  final  total  up  to  2.5  c.c. 
and  incubate  1  hour  in  water  bath.  Add  0.5  c.c.  of  5%  red  cell  suspen- 
sion and  two  units  of  amboceptor  to  each  tube  and  incubate  30  minutes. 

Tubes  2  and  3  are  necessary  control  tests  in  all  complement  fixation 
work,  to  prove  that  under  the  precise  conditions  under  which  comple- 
ment is  fixed  in  Tube  1  it  is  not  fixed  by  antigen  alone  or  by  antiserum 
alone. 

Materials  for  each  student: 

13  half-inch  test  tubes.    Test  tube  rack. 

4  large  test  tubes. 
24  hour  typhoid  culture. 
Platinum  loop. 

6  one-c.c.  pipettes. 
1.5  c.c.  immune  antityphoid  serum  (diluted). 

7.  c.c.  5  per  cent  sheep  cell  emulsion. 
4.0  c.c.  10  per  cent  guinea-pig  serum. 
30  units  amboceptor. 
Salt  solution. 


LESSON  XVII 

ALEXIN  FIXATION  BY  DISSOLVED  PROTEINS  AS  ANTIGENS 
(AND   BY   SPECIFIC   PRECIPITATES) 

THE  phenomenon  of  fixation  of  alexin  by  the  union  of  antiserum 
and  antigens  is  not  limited  to  cellular  antigens,  but  occurs  when- 
ever a  serum  containing  antibodies  is  mixed  with  its  corresponding 
protein  antigen  in  the  presence  of  alexin,  or  when  alexin  is  treated 
with  the  precipitate  so  obtained.  The  binding  of  complement 
does  not,  however,  depend  on  the  formation  of  a  precipitate,  for 
it  occurs  in  dilutions  at  which  a  precipitate  no  longer  can  be  seen. 


106 


LABORATORY  COURSE  IN  SERUM  STUDY 


Complement  fixation  is  even  more  delicate  a  method  of  identifying 
a  protein  than  is  the  precipitin  reaction. 

Reagents : 

1.  Serum  of  rabbit  immunized  against  human  serum,  diluted  1-5.1 

2.  Human  serum,  diluted  1-5. 

3.  Guinea  pig  serum,  diluted  1-10. 

4.  Washed  sheep  cells,  5  %  suspension. 

5.  Serum  of  rabbit  immunized  against  sheep  cells  (antisheep-cell 

amboceptor). 

Set  up  the  following  tubes : 


TUBE 

ANTIHUMAN  SERUM  1-5 

HUMAN-  SERUM  1-5 

Doo,  CAT,  HORSE  OR 
SHEEP  SERUM  1-5 

GuiNEA-PlQ 

SERUM  1-10 

1 

0.5  c.c. 

0.25  C.C. 

— 

0.5  c.c. 

2 

0.5  c.c. 

0.025  2 

— 

0.5  c.c. 

3 

0.5  c.c. 

0.005  2 

— 

0.5  c.c. 

4 

0.5  c.c. 

— 

0.25  c.c. 

0.5  c.c. 

5 

0.5  c.c. 

— 

— 

0.5  c.c. 

6 

— 

0.25  c.c. 

— 

0.5  c.c. 

7 

Normal  rabbit  serum 

0.25  c.c. 

— 

0.5  c.c. 

0.5  c.c. 

Bring  all  volumes  to  2  c.c. 

Incubate  mixtures  one  hour  at  37°  C. 

Sensitize  sheep  cells  by  adding  two  units  of  antisheep-cell  serum  to 
sach  0.5  c.c.  of  cells. 

At  the  end  of  the  hour  add  a  dose  of  these  sensitized  cells  to  each 
tube.  (The  dose  is  0.5  c.c.  of  5  per  cent  cells  +  the  proportionate  vol- 
ume of  amboceptor.) 

Incubate  until  haemolysis  is  complete  in  control  tubes  4,  5  and  6. 

Tube  4  is  inserted  as  a  control  test  to  show  that  fixation  is  not  given 
with  serum  in  general  but  only  with  human  serum.  Different  students 
use  the  sera  of  different  animals  as  control.  If  monkey  serum  is  avail- 
able, let  some  of  the  students  set  up  control  tests  with  it. 

Compare  the  dilution  of  human  serum  which  still  gives  a  positive 
reaction  with  the  titer  which  gave  a  precipitin  reaction  with  the  same 
antiserum  in  Lesson  VII. 

1  Where  dilutions  are  made  to  economize  material  they  must  not  bo  taken 
to  have  any  significance  of  principle. 

8  (Make  a  preliminary  dilution  of  1-20  and  add  0.5  c.c.  and  0.1  c.c.) 


108  LABORATORY  COURSE  IN  SERUM  STUDY 

MATERIALS 
Each  student  needs: 
3.0  c.c.  Antihuman  serum  (1-5). 
1.0  c.c.  Human  serum  (1-5). 
4.0  c.c.  Guinea  pig  serum  (1-10). 
4.0  c.c.  Sheep  cells  (1-20). 
0.5  c.c.  Dog,  cat,  horse  or  sheep  serum  (1-5). 
15  units  antisheep-cell  amboceptor. 

Salt  solution. 
10  one-c.c.  pipettes. 
15  half-inch  test  tubes. 

1  test  tube  rack. 

7  ^-inch  test  tubes 

3  £-inch  test  tubes 

6  1-u  pipettes. 

LESSON  XVIII 
FORENSIC   COMPLEMENT  FIXATION 

THE  fixation  of  complement  by  the  specific  action  of  a  protein 
and  the  serum  of  an  animal  immunized  against  it,  is  the  basis 
of  a  forensic  test  for  the  identification  of  blood  stains  and  also  for 
the  recognition  of  meats  placed  upon  the  market  (for  instance, 
the  detection  of  horse  meat  in  sausages). 

Since  the  injection  of  the  protein  from  any  organ  or  tissue  of 
an  animal  results  in  antibodies  which  react  with  extracts  from 
any  other  tissue  of  the  identical  species  (i.e.,  antibodies  which  are 
race-specific  and  only  to  a  slight  degree  organ-specific),  it  is 
not  necessary  to  produce  the  antiserum  for  the  detection  of  meat 
by  injection  of  meat  extract.  The  injection  of  serum  or  of  blood 
of  the  same  species  gives  quite  as  useful  antibodies  and  is  much 
more  convenient. 

For  the  preparation  of  extracts  from  meat  Seiffert  recommends 
the  infusion  of  30  grams  of  finely  chopped  meat  in  30  c.c.  salt 
solution ;  the  mixture  is  to  stand  for  12  hours  in  the  cold,  and  is 
then  filtered  through  a  hard  paper.  Students  prepare  extracts 
of  two  types  of  meat  given  out,1  extracting  5  grams  of  chopped 

1  One  of  these  should  if  possible  be  horse  meat  which  can  usually  be  ob- 
tained from  a  board  of  health  or  a  veterinary  institution.  The  antihorse  serum 
is  prepared  in  advance  by  the  injection  of  three  rabbits  with  horse  serum. 
(Lesson  I.)  If  it  is  not  possible  to  obtain  horse  flesh,  the  whole  experiment  can 
be  done  with  antisheep  serum  and  mutton  obtained  from  a  butcher. 


110 


LABORATORY   COURSE    IN   SERUM   STUDY 


meat  with  5  c.c.  of  salt  solution  and  filtering.  The  clear  solution 
represents  approximately  a  1-100  dilution  of  the  protein.  From 
it  a  1-1000  dilution  should  be  prepared. 

The  following  experiment  should  then  be  set  up  : 


TUBE 

ANTIGEN 

ANTIHOBSE  SEBUM 

GUINEA  PIG  SEBUM  1-10 

1 

Extract  A  (1-100)    0.5  c.c. 

0.1  c.c. 

0.5  c.c. 

2 

Extract  A  (1-1000)  0.5  c.c. 

0.1  c.c. 

0.5  c.c. 

3 

Extract  B  (1-100)    0.5  c.c. 

0.1  c.c. 

0.5  c.c. 

4 

Extract  B  (1-1000)  0.5  c.c. 

0.1  c.c. 

0.5  c.c. 

5 

Extract  A  (1-100)     1.0  c.c. 

— 

0.5  c.c. 

(Antigen  control) 

6 

Extract  B  (1-100)     1.0  c.c. 

— 

0.5  c.c. 

(Antigen  control) 

7 

Antiserum  control 

0.2  c.c. 

0.5  c.c. 

8 

Complement  control     — 

— 

0.5  c.c. 

All  tubes  are  to  be  brought  up  to  a  volume  of  1.5  c.c.  by  the  addition 
of  salt  solution. 

Incubate  for  one  hour  at  37.5°  C. 

At  the  end  of  this  time  0.5  c.c.  red  cells,  sensitized  with  two  units 
of  amboceptor,  are  added  to  each  tube  and  the  tubes  are  again  incubated 
until  haemolysis  is  complete  in  controls  "5,"  "6,"  "7"  and  "8." 

The  purpose  of  the  control  tubes  "5"  and  "6"  is,  of  course,  to  prove 
that  the  antigen  alone  without  the  presence  of  the  antiserum  does 
not  interfere  with  the  complement  action  of  the  guinea  pig  serum,  while 
the  purpose  of  the  control  number  "7"  is  to  make  certain  that  the 
antiserum  alone  does  not  interfere  with  the  action  of  the  complement. 
Tube  "8"  is  put  in  for  a  similar  purpose  so  that  a  slight  inhibitory 
action  (anticomplementary  effect)  on  the  part  of  either  antigen  or  anti- 
serum  can  be  recognized  by  contrast  with  the  complete  laking  which 
ensues  from  the  unhampered  effect  from  the  guinea  pig  serum  on  the 
sensitized  red  cells. 

MATERIALS 

Each  student  requires : 
5  grams  of  each  of   2   types  of 
chopped  meat  (horse  and  any 
other  variety). 

2  hard  filter  papers  and  funnels. 
1  c.c.  antihorse  serum 
5  c.c.  guinea  pig  serum  (1-10). 
20  units  antisheep-cell  amboceptor. 


8  one-c.c.  pipettes. 

8  half-inch  test  tubes. 

2  large  test  tubes  or  wide  mouth 

bottles. 

1  test  tube  rack 
5  c.c.  sheep  cells. 
Salt  solution. 


112          LABORATORY  COURSE  IN  SERUM  STUDY 

LESSON  XIX 

WASSERMANN  REACTION1 

I 

PREPARATION  OF  THE  ANTIGEN 

THE  general  principles  of  the  technique  of  the  Wassermann 
reaction  are  the  same  as  those  of  other  forms  of  complement 
fixation  already  discussed  —  with  one  notable  exception :  the 
antigen  is  not  specific  in  origin,  i.e.,  is  not  made  from  Spirochaeta 
pallida  or  (as  it  originally  was)  from  syphilitic  organs.  Land- 
steiner  and  his  successors  found  that  equally  good  or  better  re- 
sults .could  be  obtained  with  lipoidal  substances  extracted  from 
normal  organs  by  means  of  alcohol  and  other  lipoid  solvents. 
Various  lipoid  mixtures  are  now  used,  the  most  serviceable  being 
alcoholic  extracts  of  normal  heart  muscle,  —  usually  beef,  guinea 
pig,  or  human  heart.  Sometimes  cholesterin  is  added  to  the  al- 
coholic extract.  The  alcoholic  solution,  of  course,  is  well  diluted 
with  saline  solution  before  use.  These  facts,  empirically  ascer- 
tained since  the  introduction  of  the  Wassermann  reaction,  show 
definitely  that  this  test  is  not  dependent  upon  an  antigen- 
antibody  union  as  is  the  case  with  other  forms  of  complement 
fixation. 

No  matter  what  lipoid  antigen  is  used,  its  specificity  and  dos- 
age have  to  be  determined  by  careful  preliminary  titrations  and 
by  trial  on  known  syphilitic  and  non-syphilitic  sera  before  it  can 
be  used  in  diagnostic  work. 

As  the  preparation  of  antigen  for  use  in  the  Wassermann  test 
takes  time  it  had  better  be  begun  about  three  weeks  before  the 
antigen  is  needed  for  the  experiments  below.  The  preparation 
of  three  typical  and  useful  antigens  will  be  described.  The  class 
should  be  divided  into  three  groups  of  students  and  each  group 
of  students  should  be  made  jointly  responsible  for  the  prepara- 
tion of  one  or  two  specimens  of  one  of  the  types  of  antigen. 

1  Lessons  XIX  and  XX  should  be  repeated  —  if  possible,  an  entire  week 
given  to  them. 


114  LABORATORY   COURSE   IN   SERUM   STUDY 


SIMPLE   ALCOHOLIC   EXTRACT   OF   HEART   MUSCLE 

Human,  guinea  pig,  and  beef  heart  are  used  by  different  workers 
and  are  of  practically  equal  value.  Heart  muscle  is  finely  chopped 
and  extracted  at  37°  C.  in  10  volumes  of  absolute  alcohol,  for 
from  three  to  seven  days.  The  jars  in  which  the  heart  muscle  is 
kept  should  be  shaken  one  or  more  times  a  day.  If  guinea  pig 
hearts  are  used,  a  jar  of  100  c.c.  of  absolute  alcohol  is  set  aside, 
and  the  hearts  are  ground  up  and  dropped  into  the  alcohol  when- 
ever a  guinea  pig  has  to  be  killed  for  any  laboratory  purpose.  It 
is  important  to  remove  all  blood  from  the  heart.  If  beef  or  human 
heart  is  used  it  is  important  to  remove  all  fat  and  tendon  before 
the  heart  is  extracted.  At  the  end  of  the  period  of  extraction  the 
alcohol  is  filtered  and  the  filtrate  tested  as  described  below. 

B 

CHOLESTERIN   REENFORCED    HEART   EXTRACT 

The  guinea  pig  hearts 1  are  weighed  before  being  added  to  the 
alcohol.  Thirty  grams  of  finely  ground  heart  are  extracted  at 
least  14  days  at  37°  C.  with  300  c.c.  of  absolute  alcohol.  This 
is  then  filtered  and  half  the  filtrate  is  placed  in  a  flask  with  an 
excess  (say  5  grams)  of  chemically  pure  cholesterin.  This  is  kept 
at  37°  C.  overnight  and  then  in  a  water  bath  at  16°  C.  for  three 
hours  (to  precipitate  excess  of  cholesterin).  This  saturated  solu- 
tion is  then  filtered  and  the  other  half  of  the  original  heart  extract 
is  added  to  it,  giving  a  heart  extract  which  is  half  saturated  with 
cholesterin. 

•    C 

NOGUCHI'S   ACETONE-INSOLUBLE    FRACTION   OF   HEART   EXTRACT 

Whereas  in  the  preceding  antigen  cholesterin  was  added  to 
the  heart  extract,  Noguchi  obtains  a  highly  specific  antigen  by 
removing  the  cholesterin  and  certain  other  substances  from  the 
heart  extract  by  means  of  precipitation  with  acetone  (in  which 

1  Some  workers  use  human  or  beef  hearts. 


116          LABORATORY  COURSE  IN  SERUM  STUDY 

cholesterin  is  soluble  whereas  lecithin  and  certain  allied  lipoids 
are  insoluble  in  it). 

Extract  100  grams  of  mashed  heart  muscle  with  a  liter  of 
absolute  alcohol  at  37°  C.  for  several  days.  Filter  and  evaporate 
to  dryness  with  an  electric  fan.  This  usually  takes  12  to  24  hours. 
Dissolve  the  residue  in  ether  (avoid  flames).  Stand  the  milky 
ether  solution  in  the  ice  box  overnight  and  decant  off  the  clear 
supernatant  portion.  Evaporate  this  before  an  electric  fan  to  a 
small  volume  and  to  it  add  about  10  volumes  of  pure  acetone. 
Allow  to  stand  for  several  hours  and  decant  off  the  acetone. 
Scrape  the  precipitate  off  with  a  spatula  and  preserve  it  under 
acetone.  For  use  dissolve  about  0.3  grams  of  the  brownish  sticky 
precipitate  in  1  c.c.  of  ether  and  to  it  add  9  c.c.  methyl  alcohol. 
The  alcohol  solution  is  fairly  stable  and  can  be  used  as  a  stock 
solution. 

II 

TlTRATION    OF  WASSERMANN  ANTIGEN 

Alcoholic  extracts  of  organs  contain  substances  which  are 
haemolytic  and  substances  which  have  the  power  of  binding  com- 
plement non-specifically,  much  as  do  bacterial  suspensions  and 
extracts.  They  also  contain  substances  which  in  combination 
with  syphilitic  sera  have  the  power  of  binding  complement. 
It  is  important  to  test  for  all  three  of  these  properties  and  a  suit- 
able extract  must  show  an  antigenic  value  (that  is,  the  power  to 
bind  complement  in  the  presence  of  syphilitic  serum)  much  higher 
than  its  anticomplementary  or  haemolytic  values.  The  haemolytic 
power  of  the  antigen  seldom  interferes  practically,  and  is  some- 
what interfered  with  by  the  guinea  pig  and  human  serum  used  in 
the  test. 

Before  standardizing  the  antigen,  the  alcoholic  concentrated 
antigen  sliould  be  diluted  1-10  with  salt  solution,  and  the  way  in 
which  this  suspension  is  made  is  of  importance  since  the  antigen 
is  in  colloidal  suspension  and  not  in  true  solution  and  the  anti- 
complementary  and  antigenic  value  depend  certainly  in  part  on 
the  physical  condition  of  the  antigen.  This  will  vary  according 


118  LABORATORY   COURSE   IN   SERUM   STUDY 

to  whether  the  alcoholic  extract  is  run  rapidly  into  the  salt  solu- 
tion which  is  used  as  diluent  or  whether  the  extract  is  diluted  by 
dropping  salt  solution  into  it.  It  is  not  of  so  great  importance 
which  of  the  two  methods  is  used  as  that  the  same  method  should 
be  used  throughout  both  preliminary  titrations  and  tests. 

Each  student  tests  one  of  the  three  antigens  made  by  the  class. 
This  antigen  is  to  be  made  up  to  a  1-10  dilution  with  saline.  Add  the 
saline  slowly  to  the  antigen.  Each  student  needs  about  10  c.c.  of  the 
1-10  dilution. 

A  syphilitic  (Wassermann+  +  +  +)  and  a  normal  human  serum  con- 
taming  no  or  very  little  antisheep-cell  amboceptor  are  chosen :  they  are 
inactivated  at  56°  C.  for  half  an  hour  before  use. 

Set  up  the  following  series  of  tubes : 

1.  DETERMINATION  OF  ANTICOMPLEMENTARY  DOSE 

Tube  Antigen  1-10  Complement  1-10 

1  0.2  c.c.  2  units 

2  0.4  c.c.  2  units 

3  0.8  c.c.  2  units 

4  1.2  c.c.  2  units 

5  1.6  c.c.  2  units 

6  2.0  c.c.  2  units 

2.     DEMONSTRATION  THAT  ANTIGEN  is  NOT  H^MOLYTIC  OF  ITSELF 
Tube  7  Antigen  1-10  1.0  c.c.  5%  Sheep  cells  0.5  c.c. 

3.     DETERMINATION  OF  ANTIGENIC  DOSE 
Tube          Antigen         Syphilitic  (++++)  Serum  l     Complement  1-10 

8  (1-40)  0.05  c.c.  0.1  c.c.  2  units 

9  (1^0)  0.1    c.c.  0.1  c.c.  2  units 

10  (1-40)  0.2  c.c.  0.1  c.c.  2  units 

11  (1-10)  0.1  c.c.  0.1  c.c.  2  units 

12  (1-10)  0.2  c.c.  0.1  c.c.  2  units 

13  (1-10)  0.4  c.c.  0.1  c.c.  2  units 

4.  PROOF  THAT  ANTIGEN  is  SPECIFIC  (NEGATIVE  CONTROL) 

Tube  Antigen  1-10        Normal  Human  Serum  l        Complement  1-10 

14  0.1  c.c.  0.1  c.c.                               2  units 

15  0.2  c.c.  0.1  c.c.                                2  units 

16  0.4  c.c.  0.1  c.c.                               2  units 

1  The  sera  used  should  be  inactivated  on  day  of  test  by  heating  to  55°  for  twenty 
minutes  in  the  water  bath. 


120  LABORATORY  COURSE  IN  SERUM  STUDY 

5.     SERUM  CONTROLS 

Tube  Normal  Serum  Complement  1-10 

17  0.1  c.c.  2  units 

18  0.2  c.c.  2  units 

Syphilitic  (++++)  Serum 

19  0.1  c.c.  2  units 

20  0.2  c.c.  2  units 

The  volume  is  made  to  2  c.c.  as  nearly  as  possible  in  all  the  tubes. 

All  tubes  are  put  in  water  bath  for  one  hour.  Then  0.5  c.c.  of  5  per 
cent  sheep  cells  and  two  units  of  amboceptor  are  added  to  each  tube  (ex- 
cept 2  tube  7)  and  the  tubes  are  all  returned  to  the  water  bath  for 
thirty  minutes. 

CONCLUSIONS  TO  BE  DRAWN  FROM  THE  ABOVE 
Series  1 

The  anticomplementary  dose  is  that  quantity  of  antigen  which 
shows  the  slightest  inhibition  of  laking.  The  amount  of  antigen 
used  in  the  test  must  under  no  circumstances  exceed  one  half  of 
this  amount  and  is  usually  taken  as  one  third  of  it.  Thus  if 
Tube  5  containing  1.6  c.c.  of  the  1  to  10  antigen  shows  a  faint 
haze  of  undissolved  red  cells,  then  the  quantity  used  in  the  tests 
should  not  exceed  0.5  c.c. 

Series  2 

An  antigen  which  is  haemolytic  in  itself  in  Tube  7  is  rejected 
as  useless.  When  the  extractions  are  made  of  normal  heart  this 
occurrence  is  rare. 

Series  3      f 

The  antigenic  dose  is  the  smallest  amount  that  gives  com- 
plete inhibition  in  the  presence  of  syphilitic  serum.  This  is  tested 
in  Tubes  8  to  13.  In  order  that  an  antigen  be  of  value  the 
antigenic  dose  must  be  considerably  smaller  than  the  anticom- 
plementary quantity  determined  in  Series  1.  It  is  desirable  in 
the  tests  to  use  an  amount  of  antigen  containing  at  least  several 
antigenic  units.  Thus,  if  the  antigen  tested  shows  complete  in- 
hibition in  Tube  11  and  merely  partial  inhibition  in  Tube  10, 


122  LABORATORY   COURSE   IN   SERUM   STUDY 

and  perhaps  none  in  8  and  9,  then  the  quantity  contained  in 
Tube  11,  namely  0.1  c.c.  of  the  1  to  10  dilution,  is  the  antigenic 
dose  or  unit.  It  is  desirable  to  use  4  or  5  times  this  unit  in  the 
test,  and  if  this  quantity  does  not  exceed  one  third  of  the  anti- 
complementary  dose  determined  in  Series  1,  the  antigen  is  suitable 

for  use. 

Series  4 

The  object  of  Series  4  is  of  course  to  show  that  inactivated 
normal  sera  do  not  fix  complement  in  the  presence  of  the  antigen. 
In  practice  no  worker  should  be  satisfied  to  test  a  new  antigen 
against  merely  one  normal  serum,  but  before  using  the  antigen  in 
routine  tests  it  should  be  used  for  some  time  parallel  with  an  old 
familiar  antigen. 

Series  5 

Series  5  controls  the  positive  and  negative  sera  used  in  the 
preceding  series.  They  are  carried  out  because  under  certain 
circumstances  sera  become  anticomplementary,  especially  if  kept 
for  several  days.  If  this  occurs,  the  anticomplementary  property 
can  usually  be  destroyed  by  heating  to  56°  C.  However,  occa- 
sionally even  inactivated  sera  may  remain  slightly  anticomple- 
mentary. Therefore  these  controls  are  indispensable. 

MATERIALS 
Each  student  needs : 

1.0  c.c.  antigen  (undiluted). 
10.0  c.c.  complement  (1-10). 
15.0  c.c.  5  per  cent  sheep  cells. 
45  units  amboceptor. 
1.0  c.c.  positive  (+  +  +  +)  serum. 
1.0  c.c.  negative  serum. 

Salt  solution. 
25  half-inch  test  tubes. 
5  one-inch  test  tubes. 
8  one-c.c.  pipettes. 
1  two-c.c.  pipette. 
1  five-c.c.  pipette. 
1  test  tube  rack. 
Electric  fan. 


124 


LABORATORY  COURSE  IN  SERUM  STUDY 


LESSON   XX 
WASSERMANN  REACTIONS 

II 
TECHNIQUE  OF  TESTS 

t 

I.  PRELIMINARY  COMPLEMENT  TITRATION 

ON  account  of  the  complement  variability  of  guinea  pigs' 
serum  and  of  the  desirability  of  using  a  uniform  amount  of  com- 
plement, a  fresh  titration  of  the  complement  is  done  on  each 
occasion  when  Wassermann  tests  are  performed.  In  order  to 
allow  a  margin  of  safety,  twice  that  amount  of  guinea  pigs '  serum 
is  used  in  the  tests  which  in  the  preliminary  titration  gives  com- 
plete laking  with  2  units  of  amboceptor  in  15  minutes. 

(The  quantities  of  all  the  ingredients  given  here  are  known  as 
"half  Wassermann"  quantities;  that  is,  for  the  sake  of  economy  and 
convenience  half  the  amount  of  patients'  serum,  guinea  pig  serum, 
sheep  cells,  etc.,  described  in  the  original  Wassermann  test  are  used 
and  the  total  volume  is  made  to  2.5  c.c.  instead  of  5  c.c.  Some  workers 
carry  economy  farther  and  use  " tenth  Wassermann"  quantities. 

There  are  also  some  workers  who  instead  of  redetermining  the  dose 
of  complement  each  time,  keep  the  dose  of  complement  constant  — 
0.5  c.c.  of  10  per  cent  serum  for  half  Wassermanns  —  and  redetermine 
the  amboceptor  unit.  The  net  result  is  not  very  different,  but  since  the 
guinea  pig  is  the  variable  factor  it  seems  more  reasonable  to  vary  the 
amount  of  guinea  pigs'  serum.) 

Into  each  of  7  tubes  put  0.5  c.c.  of  5  per  cent  sheep  cells,  two  units 
of  amboceptor  and  10  per  cent  complement  (fresh  guinea  pig  serum)  in 
the  following  amounts: 1 


TUBE 

SHEEP  CELLS 

SENSITIZER  OR  AMBOCEPTOR 

COMPLEMENT  10%  IN  SALT 
SOLUTION 

1 

0.5  c.c. 

2  units 

0.1    c.c. 

2 

0.5  c.c. 

2  units 

0.15  c.c. 

3 

0.5  c.c. 

2  units 

0.2    c.c. 

4 

0.5  c.c. 

2  units 

0.25  c.c. 

5 

0.5  c.c. 

2  units 

0.3    c.c. 

6 

0.5  c.c. 

2  units 

0.4    c.c. 

7 

0.5  c.c. 

2  units 

0.5    c.c. 

8 

0.5  c.c. 

— 

0.5    c.c. 

Bring  the  volume  of  all  the  tubes  up  to  2.5  c.c.  by  addition  of 

saline  solution. 

1  The  following  is  the  preferable  order  of  adding  the  reagents: 

First  pipette  the  complement,  second  the  amboceptor,  third  the  saline  solution 

and  last  the  red  cells,  shaking  thf  tubes  thoroughly  while  the  cells  uro  being  :idded. 


126         LABORATORY  COURSE  IN  SERUM  STUDY 

Set  up  one  tube  (complement  control)  with  0.5  c.c.  of  sheep  cells 
and  0.5  c.c.  of  complement  only,  in  order  to  make  sure  that  the  guinea 
pig  serum  alone  is  not  at  all  haemolytic. 

For  the  tests  twice  that  amount  of  complement  is  used  which  just 
gives  complete  haemolysis  in  30  minutes.  With  average  guinea  pigs 
0.25  c.c.  gives  this  and  0.5  is  used  in  the  test. 

II.   PREPARATION  OF  SERUM 

Inactivate  at  56°  C.  for  one  half  hour  a  known  syphilitic  and  a 
known  normal  serum  and  one  or  more  sera  to  be  examined. 

III.   SETTING  UP  THE  TESTS.     PRIMARY  INCUBATION 

Test  tube  racks  with  two  rows  of  holes  are  used  so  that  each  serum 
may  have  a  " front"  tube  and  a  corresponding  "back"  tube. 

(a)  Into  each  of  a  pair  of  5  c.c.  test  tubes  measure  0.1  c.c.  of  each  of 
the  sera. 

(6)  Into  one  (the  front  tube)  of  each  pair  measure  a  dose  of  diluted 
antigen.  (The  dose  is  one  third  of  the  largest  amount  which  by 
previous  titration  has  been  found  not  to  interfere  with  the  action  of  the 
amount  of  complement  used  in  the  test.) 

Into  an  extra  tube  measure  two  doses  of  antigen  (antigen  control 
tube). 

(c)  Into  all  tubes  measure  the  predetermined  dose  of  complement. 
Shake  each  tube  at  once.     Add  enough  saline  to  make  the  contents 
of  all  the  tubes  approximately  2  c.c. 

(d)  Incubate  in  water  bath  for  one  hour  at  37°  C.     (With  certain 
antigens,  as  for  instance  with  alcoholic  heart  extracts,  somewhat  more 
delicate  results  can  be  obtained  by  a  four  hour  preliminary  incubation 
in   the   ice   box    at    8°-12°  C.      For   class   work,    however,    this    is 
impractical.) 

IV.  ADDITION  OF  ILEMOLYTIC  SYSTEM.     SECONDARY  INCUBA- 
TION.    READING  OF  RESULTS 

Into  all  tubes  measure  0.5  c.c.  of  5  per  cent  sheep  cells  to  which 
2  units  of  amboceptor  have  been  added.  Incubate  at  37°  C.  Read  the 
results  when  the  double  antigen  tube  and  all  the  serum  control  tubes 
(i.e.  those  containing  no  antigen)  are  completely  laked. 

Fill  out  a  protocol  of  the  degree  of  inhibition,  if  any,  in  the  various 
tubes : 


128 


LABORATORY  COURSE  IN  SERUM  STUDY 


INHIBITION  SHOWN 
IN  TEST 

INHIBITION  SHOWN 
IN  CONTROL  (WITH- 
OUT ANTIGEN) 

RESULT  OF 
WASSERMANN  TEST 

Positive  serum 
Negative  serum 
Unknown  serum  1 
Unknown  serum  2 
Unknown  serum  3 
Double  dose  of  antigen 

Each  student  needs: 

One  known  syphilitic  serum.          One  normal  serum. 
7  c.c.  of  10%  complement.  20  |-inch  test  tubes. 

Five  f-inch  test  tubes.        Racks.  10  one-c.c.  pipettes. 

LESSON  XXI 
COMPLEMENT  FIXATION  WITH  BACTERIAL  EXTRACTS 

ON  account  of  the  rather  high  anticomplementary  strength  of 
the  insoluble  portion  of  bacterial  bodies  themselves,  in  practical 
complement  fixation  tests  the  effort  is  made  to  obtain  the  anti- 
genie  substance  of  the  microorganisms  in  more  or  less  pure  solu- 
tion. This  is  partially  accomplished  by  extracting  with  distilled 
water,  by  mechanical  breaking  up  of  the  bacterial  bodies,  etc. 
Bacterial  complement  fixation  with  antigens  of  this  type  is  of 
practical  diagnostic  use  in  gonorrhea,  glanders,  typhoid  fever, 
whooping  cough  and  other  diseases. 

Cultures  of  the  same  species  of  organism  isolated  from  differ- 
ent sources  often  show  considerable  biological  differences,  and 
an  immune  serum  which  fixes  complement  with  one  such  strain 
often  gives  only  partial  fixation  with  another.  For  this  reason, 
in  diagnostic  tests,  polyvalent  antigens  are  used,  i.e.,  antigens 
made  of  numerous  different  strains  of  the  bacteria  in  question. 

On  account  of  the  prolonged  manipulations  required  students 
will  not  make  bacterial  antigens  for  these  tests,1  but  will  be  pro- 

1  The  technique  of  preparing  the  Gonococcus  antigen  as  practiced  at  the 
New  York  Board  of  Health  and  supplied  to  us  by  the  kindness  of  Miss  M.  P. 
Olmstead  is  as  follows : 

The  strains  used  in  the  polyvalent  antigen  are  ten  of  those  shown  by  Torrey 
to  be  serologically  distinct  —  A,  B,  C,  G,  K,  L,  N,  O,  Q,  S. 

Stock  transplants  are  kept  on  glucose  ascitic  agar,  prepared  as  follows : 
Bob  veal,  lean,  chopped  fine  1  Ib. 

Distilled  H2O  1  liter. 

Mix,  stand  overnight  at  room  tentperature.     Heat  to  45°  C.  one  hour. 


130  LABORATORY   COURSE    IN   SERUM   STUDY 

vided  with  extracts  already  made  which  require  only  to  be 
properly  diluted  with  saline  before  use,  and  with  these  extracts 
will  do  fixation  tests  for  Gonococcus  and  for  Glanders. 

Bring  to  a  boil.     Strain  through  cheese  cloth. 

Bring  to  original  volume.  Add  agar  1$  per  cent,  Witte's  peptone  2  per  cent, 
NaCl  £  of  1  per  cent. 

Titrate.     Reaction  should  be  neutral  to  phenolphthalein. 

Filter  through  a  filter  made  with  a  layer  of  cotton,  one  of  filter  paper,  another 
layer  of  cotton.  Filter  several  times. 

Autoclave  \  hour  at  15  Ib.  pressure. 

Glucose  2  per  cent. 

10  grams  of  glucose  dissolved  in  50  c.c.  distilled  H20.  Sterilize  three  days 
for  20  minutes  in  Arnold. 

Ascitic  fluid  20  per  cent. 

Ascitic  fluid  is  filtered  through  Berkefeld,  sealed  in  sterile  flasks,  incubated 
at  37.5°  several  days  before  using.  Glucose  solution  and  ascitic  fluid 
are  mixed  before  adding  to  tubed  and  sterilized  agar.  Incubate  tubes. 
Keep  two  days  before  using. 

Transplant  the  stock  cultures  every  48  hours.  The  gonococcus  must  always 
be  kept  at  37.5°  C.  Any  inequality  of  temperature  will  cause  a  poor  growth  of 
the  organism. 

For  the  antigen  transplants  use  veal  agar  prepared  as  above,  except  that  salt, 
glucose  and  ascitic  fluid  are  omitted.  The  reaction  should  be  very  carefully 
adjusted ;  it  should  be  neutral  at  the  last  titration  before  autoclaving,  and  when 
ready  to  use  0.1  to  0.2  per  cent  acid,  preferably  0.1  per  cent.  Medium  is  titrated 
hot  with  N/20  NaOH.  No  change  should  take  place  when  phenolphthalein  is 
added.  One  or  two  drops  of  NaOH  produce  a  faint  pink  color  which  disappears 
on  adding  one  or  two  drops  of  N/20  HC1.  The  agar  is  then  bottled  and  auto- 
claved  one  hah'  hour  at  15  Ib.  pressure. 

To  prepare  antigen  take  24-hour  stock  cultures  and  transplant  to  potato 
tubes  (tube  6x1  inch)  of  salt  free  veal  agar  neutral  to  phenolphthalein.  Incu- 
bate 24  hours.  Transfer  all  this  growth  by  means  of  sterile  cotton  swabs  to 
wide-mouthed  Blake  bottles  (one  tube  to  a  bottle)  containing  neutral  salt  free 
veal  agar,  gently  rubbing  the  swab  over  the  entire  surface  of  the  agar.  Incubate 
for  24  hours.  \Vash  off  all  this  growth,  if  good,  with  neutral  sterile  distilled  water, 
10  c.c.  to  a  bottle.  If  the  growth  is  poor,  5  c.c.  is  sufficient.  The  bottle  should 
be  gently  tipped  back  and  forth  two  or  three  times  after  the  water  is  added  and 
the  growth  scraped  off  lightly  with  a  bent  glass  rod.  Do  not  allow  the  water 
to  remain  on  the  agar  more  than  a  few  seconds.  The  resulting  emulsion  is  auto- 
lyzed  for  one  hour  in  a  water  bath  at  56°  C.  and  at  80°  C.  for  one  hour.  Filter 
the  autolyzed  emulsion  through  a  Buchner  funnel  which  has  been  well  packed 
with  paper  pulp  and  then  through  a  sterile  Berkefeld  filter  of  N  or  V  porosity. 

As  new  niters  are  very  alkaline  they  are  taken  to  pieces  before  use  and  boiled 
in  distilled  water  at  least  three  times,  five  minutes  each  time,  being  scrubbed 
thoroughly  with  a  small  brush  in  fresh  water  after  each  boiling.  Then  the  filter 
is  set  up  and  hot  distilled  water  allowed  to  stand  in  it  for  five  minutes.  Hot 
neutral  distilled  water  is  then  run  through  it  under  gentle  pressure  until  fluid  is 
clear  and  neutral  when  tested  with  phenolphthalein.  After  a  filter  has  been  used 


132 


LABORATORY   COURSE    IN    SERUM    STUDY 


GONOCOCCUS  COMPLEMENT  FIXATION 

The  antigen  provided  is  a  distilled-water  extract.  It  is  to  be  diluted 
1-10  with  0.9  per  cent  saline  solution.  The  preliminary  titration  is 
done  exactly  as  with  the  typhoid  emulsion  in  Lesson  XVI  in  order  to 
determine  the  "anticomplementary  dose"  of  the  antigen.  That  amount 
which  just  shows  beginning  inhibition  of  haemolysis  when  two  units  of 
amboceptor  and  two  units  of  complement  (0.5  c.c.  of  1-10  dilution)  are 
used  is  designated  the  anticomplementary  dose,  and  one  quarter  of  this 
dose  is  used  as  the  dose  of  antigen  in  the  test.  Supposing  that  this  anti- 
complementary  dose  is  found  (with  0.5  c.c.  of  1-10  guinea  pig  serum, 
0.5  c.c.  of  5  per  cent  sheep  cells,  and  two  units  of  amboceptor)  to  be  2.0 
c.c.,  then  the  tests  are  set  up  as  follows: 


TUBE 

ANTIGEN 

GUINEA  PIG 
SERUM  1-10 

1 

Positive  serum  0.1  c.c. 

0.5  c.c. 

2  units 

2 
3 

Negative  serum  0.1  c.c. 
Positive  serum  0.2  c.c. 

0.5  c.c. 

2  units 
2  units 

4 

Negative  serum  0.2  c.c. 

— 

2  units 

5 



1.0  c.c. 

2  units 

6 



2.0  c.c. 

2  units 

Bring  up  to  2.0  c.c.  with  salt  solution. 

Incubate  one  hour  in  the  thermostat  at  37°  C.  or  if  possible  four 
hours  in  the  ice  box  at  10°  C.  Then  add  0.5  c.c.  sheep  cells  sensitized 
with  two  units  of  amboceptor  and  incubate  at  37°  C.  until  all  the  tubes 
excepting  Tube  1  are  completely  laked.  The  positive  serum  is  either 
the  serum  of  a  rabbit  which  has  been  immunized  with  gonococci  or  the 
serum  of  a  patient  known  by  previous  tests  to  have  a  positive  gonococcus 
fixation  reaction.  All  the  sera  are  of  course  inactivated  at  56°  C.  for  a 
half  hour  before  use. 

it  is  boiled  in  distilled  water  and  thoroughly  scrubbed.  It  may  be  dried  in 
the  air  for  48  hours  and  put  away  or  attached  to  a  filter  flask  and  sterilized  to 
be  ready  for  use.  Do  not  use  for  filtering  gonococcus  antigen  a  filter  that  has 
been  used  for  any  other  purpose,  unless  it  is  first  boiled  in  1  per  cent  NaOH  and 
reneutralized. 

Bottle  the  filtrate  with  aseptic  precautions.  Sterilize  three  successive  days 
for  one  half  hour  each  day  at  56°  C.  and  keep  in  the  ice  box.  Immediately  before 
use  the  antigen  is  made  isotnio  by  the  addition  of  one  part  9  per  cent  saline  solu- 
tion to  nine  parts  antigen.  The  antigen  is  more  likely  to  remain  stable  if  no  salt 
is  added  until  the  day  of  use. 


134 


LABORATORY   COURSE   IN   SERUM   STUDY 


II 

GLANDERS  COMPLEMENT  FIXATION 

This  is  carried  out  in  the  same  manner  as  the  gonococcus  test, 
using  the  serum  of  a  known  glanders-infected  horse  as  a  positive  con- 
trol.1 


TUBE 

ANTIGEN 

GUINEA  PIG  SERUM  1-10 

1 

2 
3 
4 

Positive  serum    0.1  c.c. 
Negative  serum  0.1  c.c. 
Positive  serum    0.2  c.c. 
Negative  serum  0.2  c.c. 

0.5  c.c. 
0.5  c.c. 

1C)    n  ft 

0.5  c.c. 
0.5  c.c. 
0.5  c.c. 
0.5  c.c. 

c\  K  n  n 

6 



2.0  c.c. 

0.5  c.c. 

Bring  up  to  2.0  c.c.  with  salt  solution. 

Incubate  one  hour  in  the  thermostat  at  37°  C.  or  if  possible  four 
hours  in  the  ice  box  at  10°  C.  Then  add  0.5  c.c.  sheep  cells  sensitized 
with  two  units  of  amboceptor  and  incubate  at  37°  C.  until  all  tubes 
.excepting  Tube  1  are.  completely  laked.  The  positive  serum  is  from  a 
glandered  horse  known  to  be  positive  by  previous  tests. 

Materials  needed  for  each  student  for  gonococcus  fixation : 


0.5  c.c.  positive  serum. 

0.5  c.c.  normal  serum. 

5.0  c.c.  antigen  (diluted). 

5.0  c.c.  guinea  pig  serum  (1-10). 

5.0  c.c.  shesp  cells  5  per  cent  suspension. 


20  units  amboceptor. 
20  half-inch  test  tubes 

Salt  solution. 
1  test  tube  rack. 
lOone-c.c.  pipettes  (graduated). 


GLANDERS  ANTIGEN 

1  The  technique  of  preparing  glanders  antigen  as  practiced  at  the  New  York 
Board  of  Health  and  supplied  to  us  by  the  kindness  of  Miss  M.  P.  Olmstead  is  as 
follows : 

Stock  transplants  are  kept  on  glycerine  potato  agar  (for  formula  see  Park 
and  Williams'  Pathogenic  Microorganisms,  1914,  page  97)  from  1  per  cent  to 
2  per  cent  acid  to  phenolphthalein.  For  antigen  transplants  the  same  medium 
is  used  as  in  the  preparation  of  gonococcus  antigen,  except  for  the  reaction,  which 
should  be  1.6  per  cent  acid.  The  24  hour  growth  on  bottles  is  washed  off  with 
sterile,  neutral,  distilled  water  and  heated  at  80°  C.  for  6  to  8  hours,  then  filtered. 
With  the  exceptions  mentioned,  the  technique  of  antigen  preparation  is  the  same 
as  in  the  case  of  gonococcus. 


136          LABORATORY  COURSE  IN  SERUM  STUDY 

Materials  needed  for  each  student  for  glanders  fixation : 

0.5  c.c.  positive  glanders  serum  from  a  horse  known  to  be  infected 

and  giving  a  positive  glanders  complement  fixation  reaction. 
0.5  c.c.  normal  horse  serum. 
5.0  c.c.  glanders  antigen. 
5.0  c.c.  guinea  pig  serum  (1-10). 
5.0  c.c.  5  per  cent  sheep  cells. 
20  units  amboceptor. 
20  half-inch  test  tubes. 
10  one-c.c.  graduated  pipettes. 
1  test  tube  rack. 
Salt  solution. 

LESSON  XXII 
THE   STANDARDIZATION   OF  DIPHTHERIA  ANTITOXIN 

(THE  proper  understanding  of  the  work  in  this  lesson  requires 
a  review  of  the  constitution  of  diphtheria  toxins  and  the  theoreti- 
cal principles  involved.  The  student  should  read  again  the  sec- 
tion on  toxin  and  antitoxin  in  Infection  and  Resistance,  in  Kol- 
mer's  Infection  and  Immunity,  in  Paul  Th.  Miiller's  Vorlesungen 
iiber  Infektion  und  Immunitat,  in  the  Krause  und  Levaditi 
Handbuch,  or  in  some  other  work  in  which  these  principles  are 
discussed  at  length.) 

When  extensive  therapeutic  use  first  necessitated  the  estab- 
lishment of  a  standard  of  dosage  for  diphtheria  antitoxin,  it  was 
hoped  that  a  method  could  be  developed  depending  upon  simple 
in  vitro  titration  analogous  to  the  titration  of  normal  acid  against 
normal  alkali  solutions,  in  which  the  unknown  antitoxin  could 
be  measured  against  known  amounts  of  a  standard  toxin.  Since 
no  visible  or  chemically  determinable  reaction  takes  place  between 
the  two  substances  in  the  test  tube,  the  only  method  of  ascertain- 
ing whether  or  not  the  given  quantity  of  toxin  was  neutralized 
by  the  antitoxin  was  to  inject  the  two  substances,  at  first  separate 
but  later  previously  mixed,  into  susceptible  animals  and  learn 
from  the  result  whether  or  not  free,  unneutralized  toxin  was  left. 
The  guinea  pig,  chosen  as  the  most  suitable  test  animal,  therefore, 
took  the  place  in  these  reactions  of  the  "indicator"  to  demonstrate 


138          LABORATORY  COURSE  IN  SERUM  STUDY 

the  presence  of  free,  unneucralized  toxin  by  its  death,  just  as 
phenolphthalein  in.  acid-alkali  titrations  indicates  the  presence  of 
free  alkali  by  a  pink  color.  Since  guinea  pigs  of  different  weights 
and  ages  have  varying  resistances  against  the  poison,  a  standard 
weight  for  such  measurements,  namely  250  grams,  was  established. 

The  first  step  toward  such  a  standardization,  of  course,  con- 
sisted in  establishing  standards  of  measurement  for  toxin.  Beh- 
ring,  later  together  with  Ehrlich,  established  as  a  toxin  unit  or 
MLD  (minimal  lethal  dose)  the  amount  of  a  toxin  solution  which 
would  kill  a  guinea  pig  of  250  grams.  Because  of  the  importance  of 
the  time  element  this  was  later  modified  to  represent  the  amount 
which  would  kill  such  a  guinea  pig  in  from  four  to  five  days. 

The  antitoxin  unit  later  established  mainly  by  the  efforts 
of  these  two  workers  was  designated  as  the  amount  of  the  anti- 
toxin, i.e.  of  the  serum  of  a  toxin  immune  animal,  which  would 
neutralize  100  such  MLD's  (minimal  lethal  doses)  for  guinea  pigs 
of  the  standard  weight.  It  was  soon  found  by  these  workers,  as 
well  as  by  others,  that  it  was  not  easy  to  determine  the  exact 
point  of  neutralization,  that  is,  while  the  guinea  pig  might  be 
preserved  from  death  from  100  such  minimal  lethal  doses,  in  one 
case  in  another  slight  local  or  systemic  symptoms  might  easily 
escape  the  observation  of  the  investigator,  and  it  seemed  safer  to 
eliminate  the  personal  equation  entirely.  This  was  done  by  es- 
tablishing as  an  antitoxin  unit  not  the  amount  that  would  neutral- 
ize the  100  fatal  doses,  but  a  partial  neutralization  measured  in 
such  a  way  that  the  toxin  quantity  left  over  in  the  mixture  would 
still  kill  the  guinea  pig  in  four  to  five  days,  giving  the  same  effect 
as  an  unneutralized  single  toxin  unit. 

Subsequent  developments  in  the  investigation  of  toxins,  espe- 
cially by  Ehrlich,  disclosed  many  difficulties  in  the  path  of  such 
a  relatively  simple  method  of  titration.  It  is  a  comparatively 
easy  matter  to  determine  the  minimal  lethal  dose  of  any  poison 
that  one  may  have  produced.  However,  this  quantity  —  or 
MLD  —  will  not  be  identical  in  one  and  the  same  toxin  filtrate 
if  this  is  measured  at  intervals  of  a  few  weeks  or  months,  since 
the  true  toxin  is  gradually  converted  into  a  non-poisonous  prod- 


140          LABORATORY  COURSE  IN  SERUM  STUDY 

uct,  the  "toxoid."  The  toxoid  is  not  poisonous  for  guinea  pigs, 
and  therefore  the  volume  of  original  broth  necessary  to  kill  guinea 
pigs  of  the  required  weight  increases.  Now  if  this  deterioration 
into  toxoid  implied  at  the  same  time  diminution  of  neutralizing 
power  for  antitoxin  the  titration  could  be  easily  adjusted  by  simply 
remeasuring  the  minimal  lethal  dose  for  the  particular  solution 
used  and  proceeding  on  this  basis  merely  with  a  changed  unit. 
However,  in  old  toxin  solutions,  although  the  minimal  lethal 
dose  is  larger,  the  neutralizing  value  of  this  poison  for  antitoxin 
has  changed  very  little  or  not  at  all.  In  other  words,  the  toxin 
derivatives  or  toxoids,  although  no  longer  poisonous  to  the 
guinea  pig,  still  retain  their  neutralizing  power  for  the  antitoxin. 
It  is  obvious  therefore  that  no  constant  standard  could  be  obtained 
merely  by  measuring  the  minimal  lethal  dose  of  any  toxin  broth 
and  measuring  the  antitoxin  unit  against  100  such  minimal  fatal 
doses.  The  amount  of  toxoid  would  be  alike  in  no  two  toxin  filtrates 
nor  in  the  same  filtrate  at  different  times,  and  the  antitoxin  unit 
which  I  determined  against  100  MLD's  of  a  toxin  brothl  have  pro- 
duced in  my  laboratory  might  be  far  different  in  antitoxic  potency 
from  a  unit  similarly  determined  in  another  laboratory  from  an- 
other toxin  broth  containing  an  entirely  different  proportion  of 
true  toxin  and  non-poisonous  antitoxin-neutralizing  toxoids. 

In  consequence  a  different  system  has  had  to  be  worked  out 
largely  by  the  laborious  investigations  of  Ehrlich.  Ehrlich 
found  that  the  antitoxin  was  very  much  more  stable  than  the 
toxin.  If  dried,  reduced  to  a  powder,  and  preserved  in  vacuo 
in  a  small  glass  U-tube  over  phosphoric  anhydride  in  the  cold 
and  in  the  dark,  such  dried  serum  would  preserve  its  antitoxic 
value  for  a  long  time  and  no  deterioration  comparable  to  tha^  tak- 
ing place  in  the  toxin  would  occur.  Originally  Ehrlich  prepared 
tubes  in  this  way,  each  one  containing  antitoxin  powder  of  a 
potency  of  1700  antitoxin  units  to  the  gram,  these  antitoxin 
units  being  measured  against  one  of  the  toxins  in  his  possession 
representing  the  amount  necessary  to  neutralize  100  MLD's  of 
this  toxin.  Against  this  powder  then  from  time  to  time  other 
toxins  are  measured  and  the  quantity  determined  which  mixed 


142          LABORATORY  COURSE  IN  SERUM  STUDY 

with  such  an  antitoxin  unit  will  just  kill  a  guinea  pig  of  250 
grams  in  from  four  to  five  days,  i.e.  which  mixed  with  one 
standard  antitoxin  unit  will  give  the  effects  of  one  free  toxin  unit. 
This  amount  of  toxin  is  known  as  the  L  +  dose,  and  of  course 
if  such  an  L  +  dose  of  a  toxin  measured  against  the  standard  unit 
is  now  mixed  with  varying  quantities  of  an  unknown  antitoxin 
until  a  mixture  of  similar  effect  is  obtained,  the  particular  quan- 
tity of  antitoxin  used  in  this  mixture  will  be  equal  to  the  original 
standard  antitoxin  unit.  Thus  the  antitoxin  unit  established  by 
Ehrlich  is  now  the  standard  of  measurement  for  other  antitoxins 
rather  than  the  toxin  itself. 

Such  standard  antitoxin  units  are  preserved  in  the  way 
indicated  above  for  America  at  Washington,  for  Germany  in 
Frankfort,  for  France  at  Paris,  etc.,  and  the  L-j-  dose  of  toxin 
solutions  can  be  determined  against  these  standards  and  with 
such  L  +  quantities  of  any  given  toxin  new  antitoxin  can  be 
standardized  and  portions  of  these  in  turn  preserved.  Con- 
stancy of  measurement  is  thereby  assured  if  this  system  is  kept 
under  proper  governmental  supervision  without  a  break  in  con- 
tinuity, and  without  accident.  This  is  practically  insured 
against  by  the  large  number  of  laboratories  in  the  world  in  which 
this  standard  is  being  kept. 

It  is  obvious  therefore  that  in  the  investigation  of  antitoxic 
strength  three  standard  units  are  used.  They  may  be  defined 
as  follows : 

The  Minimal  Lethal  Dose  (MLD  or  T)  is  that  amount  of 
toxin  which  when  subcutaneously  injected  invariably  causes  the 
death  of  a  250-gram  guinea  pig  in  from  four  to  five  days. 

Limes  "Zero"  Dose  (L0)  is  that  amount  of  toxin  which  is  com- 
pletely neutralized  by  one  antitoxin  unit  so  that  no  trace  of  reac- 
tion, local  or  otherwise,  ensues  when  it  is  injected  mixed  with 
one  unit  of  antitoxin. 

Limes  "  Todt "  Dose  (L  -f )  is  that  amount  of  toxin  which  when 
mixed  with  one  unit  of  antitoxin  and  injected  subcutaneously 
will  cause  the  death  of  a  250-gram  guinea  pig  in  four  to  five  days. 

The  quantitative  relationship  between  the  MLD,  the  L0  dose 


144  LABORATORY   COURSE    IN    SERUM    STUDY 

and  the  L  +  dose  is  one  that  is  by  no  means  regular,  an  irregularity 
which  has  not  yet  met  with  an  entirely  satisfactory  explanation 
and  which  for  the  actual  practical  measurement  of  antitoxin 
it  is  not  necessary  to  go  into  at  present.  The  student,  however, 
is  advised  again  to  refer  to  the  textbooks  mentioned  above  and  to 
read  the  explanations  of  this  subject  carefully.  It  is  easily  seen, 
however,  that  the  definition  of  an  antitoxin  unit  as  it  is  at  present 
used  can  no  longer  be  truthfully  given  as  the  amount  which  neu- 
tralizes 100  fatal  doses  for  guinea  pigs  of  250  grams,  although  this 
is  the  way  it  is  usually  put  in  textbooks.  The  original  unit 
measured  by  Ehrlich  and  preserved  did  actually  neutralize  100 
fatal  doses  of  the  particular  toxin  used.  However,  when  the 
stated  amount  of  this  original  antitoxin  [or  of  other  antitoxins 
based  upon  it  and  similarly  preserved]  is  measured  against  many 
other  and  unknown  toxins,  the  L-h  dose  of  these  poisons  con- 
taining amounts  of  toxoid  differing  from  that  in  Ehrlich 's 
original  toxin  will  not  necessarily  contain  100  minimal  lethal 
doses  of  true  toxin.  The  antitoxin  in  such  mixtures  is  neutralized 
not  only  by  the  true  toxin  but  also  by  the  toxoid  which  has 
different  or  no  poisonous  properties,  and  thus  the  number  of 
actual  minimal  lethal  doses  contained  in  the  mixture  may  vary 
from  50  up.  An  antitoxin  unit,  therefore,  in  the  modern  sense  is 
the  amount  of  antitoxin  which  when  mixed  with  the  L  +  dose  of  a 
standard  toxin  leads  to  death  of  the  guinea  pig  in  four  to  five  days. 
The  L  +  dose  of  the  toxin  in  this  case  must  have  been  measured 
by  a  previously  determined  standard  antitoxin  unit  which  is  the 
ultimate  basis  of  measurement. 

In  order  to  allow  a  margin  of  safety  this  definition  has  been 
further  altered  of  recent  years  in  the  following  way : 

The  antitoxin  unit  is  the  amount  of  antitoxic  serum  which 
mixed  with  the  L  +  dose  of  a  standard  toxin  will  preserve  a  guinea 
pig  of  250  grams  from  death. 

PREPARATION  OF  TOXIN 

Each  student  is  given  a  500  c.c.  Erlenmeyer  flask  in  which  is  100  c.c. 
of  veal  infusion  broth  containing  2  per  cent  peptone  and  adjusted  to 


146 


LABORATORY  COURSE  IN  SERUM  STUDY 


reaction  of  0.5  per  cent  acidity  to  phenolphthalein.  (Various  modifica- 
tions of  this  medium  may  be  used.  Smith  recommends  a  broth  which 
has  been  made  sugar  free  by  inoculation  with  colon  bacillus,  and  Rosenau 
recommends  the  addition  of  1  per  cent  glucose  to  the  broth.)  A  strain 
of  B.  diphtheria  known  to  produce  strong  toxin  (preferably  the  Park- 
Williams  bacillus  No.  8)  is  used  for  inoculation.  Before  it  is  planted  on 
flasks  it  should  be  transferred  by  the  instructor  daily  through  a  series 
of  several  broth  tubes  to  obtain  vigorous  and  rapid  growth.  The  flasks 
are  incubated  for  seven  days  and  one  or  two  which  show  the  best  growth 
filtered  through  a  Berkefeld  filter.  The  filtrate  is  stored  in  a  sterile 
dark  bottle  on  ice  until  used. 

The  class,  working  as  a  group,  will  carry  out  one  or  more  of  the 
following  determinations  on  one  such  toxin,  each  student  injecting  one 
of  the  pigs  in  a  given  series.  (In  actual  class  work,  in  order  to  save 
guinea  pigs,  usually  only  III,  the  determination  of  the  antitoxic  value 
of  an  unknown  serum,  is  carried  out.) 

I.    DETERMINATION   OF   THE   MLD   OF   A  TOXIN 

Guinea  pigs  are  injected  subcutaneously  with  varying  amounts 
of  this  toxin  until  the  amount  is  determined  which  will  regularly 
cause  death  in  from  four  to  five  days.  For  a  satisfactory  toxin  the 
MLD  must  be  less  than  0.01  c.c.  The  toxin  is  diluted  in  salt  solution 
so  that  the  desired  amount  will  be  contained  in  2  c.c.  This  amount  is 
injected  subcutaneously,  with  a  Rosenau  syringe,1  the  needle  of  which 
is  inserted  in  the  flank  of  the  guinea  pig  and  passed  subcutaneously  in 
the  flank  of  the  pig  till  the  point  is  near  the  linea  alba  before  the  diluted 
poison  is  injected.  After  injection  1  c.c.  of  salt  solution  is  placed  in 
the  syringe  without  removing  the  needle  and  injected  to  wash  out  the 
last  traces  of  the  poison.  The  following  example  of  such  a  determination 
is  taken  from  Rosenau's  toxin  No.  5.  The  MLD  in  this  case  was  .002  c.c. 


DATES  TESTED 

ANIMAL 

TOXIN  INJECTED 

RESULT 

5/26/04 

G.P.        #9 

.005    O.C. 

Death  1  day    18  hours 

5/26/04 

G.P.      #  10 

.005     C.C. 

Death  1  day   20  hours 

5/31/04 

G.P.      #  13 

.004    c.c. 

Death  3  days  12  hours 

5/31/04 

G.P.      #  12 

.003    c.c. 

Death  2  days  12  hours 

5/31/04 

G.P.      #11 

.002    c.c. 

Death  4  days    0  hours 

7/21/04 

G.P.    #105 

.001    o.c. 

Death  5  days    5  hours 

7/21/04 

G.P.    #107 

.0008  o.c. 

Death  7  days  23  hours 

9/30/04 

G.P.    #311 

.002    c.c. 

Death  4  days    3  hours 

12/15/04 

G.P.  #  1030 

.002    c.o. 

Death  4  days    8  hours 

1  Ordinary  Luer  or  other  all-glass  syringes  are  used  in  many  laboratories. 


148 


LABORATORY   COURSE   IN   SERUM   STUDY 


II.    DETERMINATION   OF   THE   L+    DOSE   OF   TOXIN 

A  dilution  of  a  carefully  standardized  antitoxic  serum  is  prepared 
in  sterile  salt  solution  so  that  each  cubic  centimeter  contains  one  unit 
of  antitoxin.  A  preliminary  test  with  the  dilutions  spaced  widely 
should  be  carried  out  by  the  instructor  to  determine  approximately 
the  L+  dose.  A  series  of  dilutions  of  toxin  increasing  by  about  TV 
of  the  smallest  amount  should  then  be  prepared,  the  total  volume  of  each 
dilution  being  brought  up  to  2  c.c.  (The  smallest  amount  is  the  amount 
just  below  the  minimal  dose  fatal  when  mixed  with  one  unit  of  antitoxin 
in  the  preliminary  experiment.)  1  c.c.  of  antitoxin  is  then  mixed  with 
2  c.c.  of  each  dilution  of  toxin  in  a  Rosenau  syringe  and  allowed  to  stand 
for  one  hour.  The  mixture  is  then  injected  into  a  250-gram  pig,  as  in 
determining  the  MLD,  and  the  syringe  washed  with  1  c.c.  of  salt  solution. 

If  the  preliminary  test  has  shown  the  dose  to  be  between  0.2  and  0.3 
c.c.  the  test  should  be  set  up  as  follows : 


TUBE 

ANTITOXIN 

TOXIN  (1  IN  5) 

SALT  SOLXJTION 

1 

1.0  c.c.  (=  1  unit) 

1.0  c.c. 

1.0  c.c. 

2 

1.0  c.c. 

1.1  c.c. 

0.9  c.c. 

3 

1.0  c.c. 

1.2  c.c. 

0.8  c.c. 

4 

1.0  c.c. 

1.3  c.c.  . 

0.7  c.c. 

5 

1.0  c.c. 

1.4  c.c. 

0.6  c.c. 

6 

1.0  c.c. 

1.5  c.c. 

0.5  c.c. 

If  the  pigs  injected  with  the  last  two  mixtures  die  on  or  before  the 
fourth  day,  and  those  injected  with  the  first  four  mixtures  survive  but 
possibly  develop  late  paralysis,  or  if  they  die  after  the  fifth  day,  the  L+ 
dose  is  1.4  c.c.  of  1  in  5  toxin,  or  0:28  c.c. 


III.    DETERMINATION    OF    THE    ANTITOXIC    VALUE    OF    AN    UNKNOWN 

SERUM 

The  L+  dose  of  a  toxin  having  been  carefully  determined,  this  toxin 
may  be  used  for  standardizing  an  unknown  antidiphtheritic  serum  by  an 
analogous  procedure.  In  this  case  the  toxin  is  diluted  so  that  2  c.c. 
contain  precisely  the  L+  dose.  Preliminary  tests  should  be  made  to 
determine  approximately  the  unit.  Then  an  accurate  test  is  carried 
out  as  follows : 

If  the  serum  is  found  in  the  preliminary  determination  to  contain 
between  200  and  400  units  per  c.c.  the  dilutions  are  made  as  follows : 
A  stock  dilution  of  1  c.c.  of  serum  plus  19  c.c.  of  salt  solution  is  prepared 


150 


LABORATORY  COURSE  IN  SERUM  STUDY 


and  the  following  series  of  dilutions  accurately  made  from  this  in  test 
tubes : 


TUBE 

ANTITOXIN 

SALT  SOLUTION 

DILUTION 

1 

1.0  c.c. 

9.0  c.c. 

1-200 

2 

1.0  c.o. 

10.0  c.c. 

•  1-220 

3 

1.0  c.c. 

11.0  c.c. 

1-240 

4 

1.0  c.c. 

12.0  c.c. 

1-260 

5 

1.0  c.c. 

13.0  c.c. 

1-280 

6 

1.0  c.c. 

14.0  c.c. 

1-300 

7 

1.0  c.c. 

15.0  c.c. 

1-320 

8 

1.0  c.c. 

16.0  c.c. 

1-340 

9 

1.0  c.c. 

17.0  c.c. 

1-360 

10 

1.0  c.c. 

18.0  c.c. 

1-380 

11 

1.0  c.c. 

19.0  c.c. 

1-100 

1  c.c.  of  each  dilution  is  then  mixed  in  a  Rosenau  syringe  with  2  c.c. 
of  diluted  toxin,  the  mixture  allowed  to  stand  one  hour  at  room  tempera- 
ture, injected  subcutaneously  into  a  guinea  pig  in  the  manner  described 
for  determining  the  MLD,  the  syringe  washed  with  salt  solution  and  the 
pigs  numbered  and  carefully  observed.  Should  pigs  receiving  dilutions 
1  to  3  survive  for  five  days,  and  the  pigs  receiving  the  higher  dilutions 
die  in  two  to  five  days  the  serum  in  question  would  contain  260  units 
per  c.c.  Guinea  pigs  which  die  should  be  autopsied  and  the  character- 
istic lesions  found. 

To  provide  a  margin  of  safety  in  testing  commercial  sera  it  is  cus- 
tomary to  consider  the  smallest  unit  which  serves  to  protect  the  pig  as  a 
unit,  in  this  case  -^  of  1  c.c.,  which  means  that  there  are  240  units 
to  the  cubic  centimeter. 

Materials  needed  for  III,  Determination  of  unknown  antitoxin  : 

Antitoxin.  , 

Diphtheria  toxin  whose  M.  L.  Dose  and  L-f  Dose  are  exactly  known. 

Precision  syringes. 

12  guinea  pigs  of  approximately  250  grams  weight. 

LESSON  XXIII 

TETANUS   TOXIN 

TETANOLYSIN  AND  ANTITETANOLYSIN 

MANY  bacteria,  for  instance  tetanus  bacillus,  Staphylococcus, 
various  vibrios,  Bacillus  megatherium  and  Bacillus  proteus, 
develop,  in  culture  media,  substances  which  are  capable  of  laking 
red  cells.  These  substances  are  relatively  thermolabile  and 


152 


LABORATORY  COURSE  IN  SERUM  STUDY 


deteriorate  readily  in  solution.  They  are  known  as  haemotoxins 
and  their  injection  in  suitable  doses  into  animals  leads  to  the  pro- 
duction of  antitoxins  whose  specific  protective  action  can  be 
demonstrated  in  vitro. 

These  hsemotoxins  also  have  a  hsemolytic  action  in  the  body, 
although  they  play  a  smaller  role  than  the  other  toxic  components 
(neurotoxin,  for  instance,  in  the  case  of  tetanus).  Their  in  vivo 
hsemolytic  effect  is  probably  greatly  hindered  by  the  protective 
power  which  normal  serum  possesses  when  in  sufficient  concen- 
tration. 

I.  DETERMINATION  OF  MINIMAL  LAKING  DOSE  OF  TETANUS  TOXIN 

Tetanus  toxin  (either  a  recently  prepared  filtrate  or  a  .toxin  precipi- 
tated with  ammonium  sulphate  and  preserved  in  the  dry  state  and  made 
up  to  1  per  cent  solution  in  0.9  per  cent  saline) l  is  tested  as  follows  : 


TUBE 

TOXIN  SOLUTION 

5  %  RABBIT  CELLS 

SALINE 

1 

0.5    c.c. 

0.5  c.c. 

1.0    c.c. 

2 

0.2    c.c. 

0.5  c.c. 

1.3    c.c. 

3 

0.1    c.c. 

0.5  c.c. 

1.4    c.c. 

4 

0.05  c.c. 

0.5  c.c. 

1.45  c.c. 

5 

0.0 

0.5  c.c. 

1.5    c.c. 

Incubate  one  hour  at  37°  C.,  and  observe  haemolysis. 

II.    TlTRATION  OF  ANTITETANOLYSIN  IN   IMMUNE   HORSE   SERUM 

(TETANUS  ANTITOXIN) 


TUBE 

TOXIN 

IMMUNE  SERUM 

SALINE  TO  MAKE  VOLUMES 

UP  TO   1.5  C.C. 

1 
2 
3 
4 
5 

One  minimal  laking  dose 

M 

«i 
M 
It 

1-1000        1.0  c.c. 
1-1000        0.5  c.c. 
1-10,000     1.0  c.c. 
1-10,000     0.5  c.c. 
1-10,000     0.2  c.c. 

i 

i 
t 

i 

NORMAL  HORSE  SERUM 

6 

7 
8 
9 

it 

M 
it 

No  toxin 

1-100          1.0  c.c. 
1-1000        1.0  c.c. 
No  serum 
1-100          1.0  c.c. 

it 
tt 
ii 
it 

1  A  preliminary  trial  by  the  instructor  is  desirable  before  the  toxin  is  given 
out  to  the  class,  as  the  quantities  of  toxin  given  in  the  table  may  have  to  be 
modified. 


154          LABORATORY  COURSE  IN  SERUM  STUDY 

After  one  half  hour  at  37°  C.  add  0.5  c.c.  of  5  per  cent  rabbit  cells 
to  all  tubes,  incubate  one  hour,  and  observe  haemolysis. 

Tube  8  is  inserted,  of  course,  to  make  certain  that  the  toxin  does 
not  lose  its  haemolytic  power  during  the  half  hour  of  preliminary  incuba- 
tion, and  Tube  9  to  show  that  horse  serum  is  not  of  itself  haemolytic. 

TETANOSPASMIN 

Students  should  work  in  groups  of  three  or  four  in  performing 
the  following  experiments.  Fresh  tetanus  toxin  is  obtained  from 
a  department  of  health  or  one  of  the  commercial  laboratories  with 
the  lethal  dose  for  guinea  pigs  of  350  grams  worked  out.  Tetanus 
antitoxin  can  be  procured  from  the  same  source  or  in  the  market. 

A.  Inject  a  guinea  pig  of  approximately  350  grams  into  the  thigh  of 
one  hind  leg  with  five  lethal  doses  of  the  tetanus  toxin. 

B.  A  similar  guinea  pig  at  the  same  time  is  injected  with  twice  the 
dose,  i.e.  ten  lethal  doses  of  tetanus  toxin,  which  has  been  mixed  with 
one  antitoxin  unit,1  the  mixture  having  been  allowed  to  stand  at  room 
temperature  for  twenty  minutes. 

C.  Grind  up  five  lethal  doses  of  the  tetanus  toxin  with  half  of  one 
cortex  of  fresh  guinea  pig  brain,  adding  salt  solution  to  the  amount  of 
3  or  4  c.c.  during  the  process.     This  mixture  is  allowed  to  stand  at 
room  temperature  for  two  or  three  hours.     It  is  then  centrifugalized 
and  the  supernatant  fluid  injected  into  a  third  pig. 

The  pigs  are  kept  under  close  observation  for  6  days. 

Materials  required  for  each  student  for  the  tetanolysin  ex- 
periments:      Tetanus  toxin  about  4     c.c. 
Tetanus  antitoxin  about  0.1  c.c. 
Normal  horse  serum  about  0.1  c.c. 
5  per  cent  rabbit-cell  suspension  8.0  c.c. 

Materials  for  each  group  of  3  or  4  students  for  the  tetanospas- 
min  experiment : 

3  guinea  pigs  of  about  350  gms.  each. 

20  lethal  doses  of  tetanus  toxin  of  known  toxicity. 

Tetanus  antitoxin,  one  unit. 

J  fresh  guinea  pig  brain. 

1  The  antitoxin  unit  is  established  by  the  United  States  Hygienic  Laboratory 
(Bulletin  43)  on  principles  similar  to  thos«-  u-« •<!  for  diphtheria  antitoxin  and 
represents  ten  times  the  amount  of  antitoxin  necessary  to  completely  neutralize 
100  minimal  lethal  doses  of  a  standard  toxin  injected  into  a  guinea  pig  of  350 
grams. 


156 


LABORATORY  COURSE  IN  SERUM  STUDY 


LESSON  XXIV 
TOXINS   OF  HIGHER  PLANTS  AND   ANIMALS 

MANY  of  the  higher  plants  and  animals  produce  toxins  resem- 
bling in  every  way  the  toxins  of  bacterial  origin.  The  property 
which  is  essential  for  the  identification  of  such  substances  as  toxins 
is  the  possibility  of  producing  antitoxins  for  them.  The  other 
properties,  such  as  the  extraordinary  potency  and  thermolability, 
are  less  essential.  Most  of  them  have  more  than  one  toxic  com- 
ponent, like  many  of  the  bacterial  toxins  (for  instance,  tetanus 
toxin).  Thus  beside  the  effect  on  the  nervous  system  and  blood 
vessels  snake  poisons  are  hsemolytic,  and  ricin,  a  powerful  toxin 
derived  from  the  castor  bean,  is  hsemagglutinative. 

Human  and  guinea  pig  cells  are  susceptible  to  laking  by 
cobra  venom  in  the  absence  of  serum  or  lecithin.  Sheep  and  ox 
cells  are  highly  resistant.  The  addition  of  lecithin  or  of  suitable 
serum,  however,  makes  the  insusceptible  cells  again  susceptible 
to  laking  by  the  venom. 

Kyes  at  first  supposed  that  the  lecithin  "  activated  "  the  hsemo- 
lysin  of  the  cobra  venom  by  an  action  analogous  to  that  of  com- 
plement in  the  case  of  the  specific  hsemolysins.  Later  researches, 
especially  those  of  von  Dungern  and  Coca,  showed  that  the  venom 
contained  a  lipoid-splitting  enzyme  or  lipase  which  acted  upon  the 
lecithin  of  the  cell  membrane  or  the  lecithin  contents  of  the  serum 
and  liberated  split  products  which  possessed  haemolytic  action. 

Set  up  the  following  tests  : 

HjEMOLYTTC    EFFECT   OF    COBRA   VENOM 


TUBE 

RED  BLOOD  CELLS 

GUINEA  Pio  SERUM  OR 
LECITHIN  EMULSION 
1-2000 

COBRA  VENOM 
1-1000 

SALT  SOLUTION 

1 

Guinea  pig  cells 

5%  0.5 

— 

0.1  c.c. 

0.9  c.c. 

2 

Sheep  cells 

5%  0.5 

— 

0.1  c.c. 

0.9  c.c. 

3 

Sheep  cells 

0.5 

G.  P.  0.1  c.c. 

0.1  c.c. 

0.8  c.c. 

4 

Sheep  cells 

0.5 

L.       0.4  c.c. 

0.1  c.c. 

0.5  c.c. 

5 

Sheep  cells 

0.5 

G.  P.  0.1  c.c. 

— 

0.9  c.c. 

6 

Sheep  cells 

0.5 

L.       0.4  c.c. 

— 

0.6  c.c. 

Incubate  one  hour  at  37°  C.,  making  observations  every  15  minutes, 
and  note  whether  the  occurrence  of  haemolysis  confirms  the  statements 
made  above. 


158  LABORATORY   COURSE   IN   SERUM   STUDY 

HwEMAGGLUTINATIVE   EFFECT   OF   RlCIN 

Ricin  agglutinates  the  cells  of  all  species  to  a  greater  or  lesser 
degree.  The  presence  of  serum  in  any  considerable  quantity 
interferes  with  the  reaction.  Specific  antitoxic  sera  (which  are 
difficult  to  prepare)  neutralize  in  high  dilutions  not  only  the 
poisonous  but  also  the  agglutinative  component  of  ricin. 


TUBE 

GUINEA  Pio  CELLS  5% 

RICIN  1-1000 

SHEEP  SERUM 

1 

0.5  C.C. 

0.1  c.c. 



2 

0.5  c.c. 

0.5  c.c. 

— 

3 

0.5  c.c. 

1.0  c.c. 

— 

4 

0.5  c.c. 

0.1  c.c. 

1.0  c.c. 

5 

0.5  c.c. 

0.5  c.c. 

1.0  c.c. 

6 

0.5  c.c. 

1.0  c.c. 

1.0  c.c. 

Incubate  one  hour. 


Make  all  volumes  equal  by  adding  saline. 
Observe  hsemagglutination. 

Each  student  requires : 
5.0  c.c.  guinea  pig  cells  5  %. 
0.5  c.c.  guinea  pig  serum. 
7.0  c.c.  sheep  cells. 
8.0  c.c.  sheep  serum. 
1.0  c.c.  lecithin  diluted  1-2000. 
1.0  c.c.  cobra  venom  diluted  1-1000. 
4.0  c.c.  ricin  diluted  1-1000. 
Salt  solution. 

LESSON  XXV 


DETERMINATION    OF    ANTITRYPTIC    ACTIVITY    OF    BLOOD 

SERUM 

NORMAL  blood  contains  a  trypsin-like  ferment.  This  enzyme 
digests  protein  in  neutral  or  weakly  alkaline  solution,  with  the 
formation  of  amino-acids.  Under  ordinary  conditions  its  action 
is  inhibited  by  the  presence  of  substances  in  serum  which  have 
been  shown  by  Jobling  and  Peterson  to  be  compounds  of  unsat- 
urated  fatty  acids. 

This  antitryptic  activity  is  greatly  increased  in  cachectic 


160         LABORATORY  COURSE  IN  SERUM  STUDY 

individuals  and  tests  for  increase  in  antitrypsin  have  been  pro- 
posed as  an  aid  to  diagnosis  of  carcinoma.  The  condition,  how- 
ever, is  not  limited  to  patients  with  malignant  diseases,  but  is 
present  also  in  pernicious  anaemia,  in  Graves'  disease,  in  ad- 
vanced tuberculosis,  and  in  many  other  infections. 

A  specific  antitrypsin  which  may  be  quite  different  in  its  nature 
from  the  substance  present  in  normal  serum  is  developed  in  re- 
sponse to  injections  of  trypsin  into  animals.  The  precise  deter- 
mination of  the  antitryptic  activity  of  the  serum  in  experimental 
work  is  made  by  adding  serum  to  mixtures  of  trypsin  and  some 
protein  substrate  and  after  incubation  comparing  the  amount  of 
incoagulable  nitrogen  in  the  flasks  containing  serum  with  the 
amount  in  control  flasks  containing  trypsin  alone. 

Simpler  methods  have  been  devised  for  clinical  use.  One 
widely  used  consists  in  placing  drops  of  mixtures  of  trypsin  and 
serum  in  varied  proportion  on  the  surface  of  Loeffler's  blood  serum 
plates  and  determining  the  action  of  the  trypsin  by  the  formation 
of  a  pit  on  the  surface  of  the  plate  underneath  the  drop  of  fluid. 

A  more  satisfactory  method  is  that  of  Fuld  and  Gross  as 
follows :  Mixtures  of  serum  with  varying  concentrations  of  tryp- 
sin are  allowed  to  act  on  an  alkaline  solution  of  casein  and  after 
incubation  the  undigested  casein  is  precipitated  by  the  addition 
of  acetic  acid. 

Reagents : 

1.  Trypsin  solution.     Dissolve  0.5  gram  of  trypsin  in  50  c.c.  of 
salt  solution  containing  0.5  c.c.  of  normal  soda.     Make  up  to  500  c.c. 
with  salt  solution.     Samples  of  commercial  trypsin  vary  greatly  in  their 
activity  and  this  stock  solution  should  be  diluted  if  necessary  until  the 
amount  required  to  digest  2  c.c.  of  casein  solution  is  approximately 
0.5  c.c. 

2.  Casein  solution.     Dissolve  1  gram  of  casein  in  100  c.c.  of  N/10 
NaOH  by  warming  and  neutralize  the  solution  with  N/10  HC1,  using 
litmus  as  an  indicator.     The  volume  is  brought  up  to  500  c.c.  with  salt 
solution  and  the  solution  filtered.     It  should  be  sterilized  in  the  Arnold 
sterilizer  if  it  is  not  to  be  used  immediately. 

3.  Acetic  acid  solution  composed  of  glacial  acetic  acid  5  c.c., 
alcohol  45  c.c.,  water  50  c.c. 


162 


LABORATORY   COURSE    IN   SERUM   STUDY 


A.     TlTRATION   OF   TRYPSIN    SOLUTION 

In  each  of  a  series  of  test  tubes  place  decreasing  amounts  of  trypsin 
solution,  1  c.c.,  0.7,  0.5,  0.4,  0.3  and  0.2  c.c.  Add  sufficient  salt  solution 
to  bring  up  the  volumes  to  2.5  c.c.  and  set  up  a  control  containing  2.5  c.c. 
of  saline  alone.  To  each  tube  add  2  c.c.  of  casein  solution,  shake,  and 
place  in  water  bath  for  half  an  hour  at  37°  C.  Remove  the  tubes  from 
the  water  bath  and  add  3  to  4  drops  of  the  acetic  acid  solution  to  each. 
The  tube  with  the  smallest  amount  of  trypsin  which  shows  no  precipi- 
tate of  undigested  casern  contains  the  amount  of  trypsin  needed  for 
the  subsequent  test. 

PRELIMINARY  TITRATION  OF  TRYPSIN 


TUBE 

TBYPSIN  SOLUTION 

SALINE 

CASEIN  SOLUTION 

RESULT 

1 

1.0  c.c. 

1.5  c.c. 

2.0  c.c. 

2 

0.7  c.c. 

1.8  c.c. 

2.0  c.c. 

3 

0.5  c.c. 

2.0  c.c. 

2.0  c.c. 

4 

0.4  c.c. 

2.1  c.c. 

2.0  c.c. 

5 

0.3  c.c. 

2.2  c.c. 

2.0  c.c. 

6 

0.2  c.c. 

2.3  c.c. 

2.0  c.c. 

7 

0.0  c.c. 

2.5  c.c. 

2.0  c.c. 

B.   DETERMINATION  OF  ANTITRYPTIC  ACTIVITY  OF  SERUM 

For  control  a  mixture  of  several  normal  sera  is  used  and  a  2  per  cent 
solution  in  normal  saline  is  prepared.  A  2  per  cent  solution  of  the  serum 
to  be  tested,  preferably  from  a  case  of  advanced  carcinoma,  is  used  for 
the  test. 

In  order  to  demonstrate  the  lipoidal  nature  of  normal  antitrypsin  it 
can  be  removed  from  serum  by  chloroform  extraction.  4  c.c.  of  diluted 
normal  serum  should  be  placed  in  a  test  tube,  0.5  c.c.  of  chloroform  added, 
the  mixture  thoroughly  shaken  and  incubated  for  one  hour,  at  the  end 
of  which  time  the  mixture  is  centrifugalized  and  the  clear  supernatant 
fluid  pipetted  off.  The  normal  serum,  the  carcinoma  serum,  and  the 
normal  serum  after  chloroform  extraction  should  be  tested  in  parallel 
as  follows : 

In  each  of  six  tubes  place  0.5  c.c.  of  -the  2  per  cent  solution  of  serum. 
To  the  first  tube  add  the  dose  of  trypsin  which  will  completely  digest  the 
casein  as  determined  by  previous  titration  (this  is  "the  unit"  of  trypsin), 
and  in  the  successive  tubes  increasing  amounts  of  the  trypsin  so  that 
Tube  6  contains  four  times  as  much  trypsin  as  Tube  1.  For  example,  if 


164 


LABORATORY   COURSE   IN   SERUM   STUDY 


in  the  preliminary  test  the  tube  containing  0.5  c.c.  of  trypsin  solution 
was  completely  dissolved  this  series  of  tubes  should  contain  0.5,  0.75,  1.0, 
1.25,  1.5  and  2.0  c.c.  respectively.  Sufficient  salt  solution  is  added  to 
bring  the  volumes  up  to  2.0  c.c.,  and  2.0  c.c.  of  casein  solution  added 
to  each  tube.  The  tubes  are  incubated  for  one  half  hour  and  at  the  end 
of  this  time  the  undigested  casein  is  precipitated  by  the  addition  of  a 
few  drops  of  the  acetic  acid  solution. 

In  the  presence  of  carcinoma  serum  from  1J  times  to  twice  the 
amount  of  trypsin  will  be  found  necessary  to  completely  digest  the 
casein  that  is  necessary  in  the  presence  of  normal  serum.  On  the  other 
hand,  in  the  serum  which  has  been  extracted  with  chloroform  the  anti- 
tryptic  activity  will  be  found  to  have  been  almost  completely  removed. 

TESTING  OF  SERUM 


TUBE 

TRYPSIN 
SOLUTION 

PATIENT'S 
SERUM  —  2% 

SALINE  TO  BRING 
VOLUMES  UP  TO  2  c.c. 

CASEIN 
SOLUTION 

RESULT 

1 

1    unit 

0.5  c.c. 

it 

2  C.C. 

2 

li  units 

0.5  c.c. 

« 

2  c.c. 

3 

2   units 

0.5  c.c. 

2  c.c. 

4 

2|  units 

0.5  c.c. 

2  c.c. 

5 

3    units 

0.5  c.c. 

2  c.c. 

6 

4   units 

0.5  c.c. 

2  c.c. 

7 

None 

0.5  c.c. 

2  c.c. 

LESSON  XXVI 
NATURE  OF  NORMAL  OPSONIN 

INTRODUCTORY  REMARKS 

IN  the  early  days  of  immunological  investigation  it  was  sup- 
posed that  phagocytosis  depended  upon  properties  inherent  in 
the  phagocyting  cell.  More  recent  investigations,  starting  with 
those  of  Metchnikoff,  continued  by  Denys  and  Leclef  and  others, 
and  brought  to  focus  by  Wright  and  his  associates,  have  shown 
that  in  phagocytosis,  at  least  when  carried  on  by  the  polynuclear 
cells  of  the  circulating  blood,  it  is  a  function  of  the  cooperation 
of  serum  and  phagocyte.  It  has  been  found  that  the  serum 
so  acts  upon  the  bacteria  that  they  are  rendered  more  easily 


166          LABORATORY  COURSE  IN  SERUM  STUDY 

taken  up  by  the  leucocytes,  and  that  the  serum  constituent 
which  carries  out  such  preparatory  action  is  comparable  to  other 
antibodies.  The  property  of  the  serum  which  carries  out  this 
preparation  of  the  bacteria  for  phagocytosis  has  been  spoken  of 
by  Wright  as  the  opsonic  power,  since  he  supposed  it  to  depend 
upon  a  specific  antibody,  which  he  called  opsonin.  In  immune 
sera  this  phagocytosis-aiding  antibody  is  more  heat-resistant 
than  it  is  in  normal  sera,  and  such  a  heat-resistant  immune  body 
is  spoken  of  by  the  Wright  school  as  the  immune  opsonin  and  by 
Neufeld  and  his  associates  as  bacteriotropin. 

Serum.  —  Obtain  normal  guinea  pig  serum  by  bleeding  two  pigs 
from  the  carotid  into  centrifuge  tubes  and  separating  serum. 

Leucocytes.  —  Wash  out  peritoneum  of  two  guinea  pigs  injected  in- 
traperitoneally  with  6  c.c.  of  aleuronat  suspension  l  about  8  to  12  hours 
before.  Take  2  c.c.  of  suspension,  centrifugalize,  wash  the  sediment 
once  in  saline  and  suspend  in  saline  until  emulsion  corresponds  in  thick- 
ness to  standard  tube  prepared  by  instructor. 

Bacteria.  —  Make  suspension  of  staphylococcus  aureus  from  agar 
culture  in  salt  solution  until  thickness  corresponds  to  standard  tube. 
Draw  in  and  out  of  capillary  pipette  until  the  bacteria  are  evenly  emulsi- 
fied. Allow  clumps  to  settle  and  pipette  off  upper  layers. 

Experiment  1 

OPSONIC  ACTION  OF  SERUM 
Set  up  three  tubes  as  follows: 


TUBE 

GUINEA  Pio  SERUM 

SUSPENSION  OP  BACTERIA 

SUSPENSION  OF  LEUCOCYTES 

1 
2 
3 

Fresh                0.25  c.c. 
(Inactivated)   0.25  c.c. 
Salt  solution    0.25  c.c. 

0.1  C.C. 

0.1  c.c. 
0.1  c.c. 

0.2  c.c. 
0.2  c.c. 
0.2  c.c. 

All  tubes  are  incubated  one  half  hour  at  37°  C.  Two  smears  are 
then  made  of  the  sediment  on  slides  and  stained  by  Gram's  method, 
or  with  Jenner's  stain.  To  obtain  material  for  smears  pour  out  the 
supernatant  fluid  and  scrape  the  leucocytes  from  the  bottom  of  the  tube 
with  a  platinum  loop.  The  bacteria  in  a  hundred  consecutive  leucocytes 
on  each  slide  are  then  counted. 

1  To  make  aleuronat  suspension  take 

Aleuronat  5 

Starch  3 

Water  or  broth        100 

Mix  cold.    Bring  to  a  boil.    Tube  and  sterilize. 
For  washing  out  the  guinea  pig's  abdomen  use  sod.  citrate  solution  in  saline. 


168          LABORATORY  COURSE  IN  SERUM  STUDY 

Experiment  2 

THE  OPSONIN  UNITES  WITH  THE  BACTERIA,  NOT  WITH  THE 
LEUCOCYTES 

In  two  small  centrifuge  tubes  mixtures  are  made  with  the  same  ma- 
terials employed  in  Experiment  1,  as  follows : 

(1)  Bacterial  suspension  0.1  c.c. 
Fresh  guinea  pig  serum  0.2  c.c. 
Salt  solution  0.7  c.c. 

(2)  Leucocyte  suspension  0.2  c.c. 
Fresh  guinea  pig  serum  0.2  c.c. 
Salt  solution  0.6  c.c. 

These  tubes  are  allowed  to  stand  at  37°  C.  in  water  bath  or  incu- 
bator for  30  minutes.  Then  centrifugalize  vigorously  and  wash  sedi- 
ment once  in  salt  solution. 

To  the  bacterial  sediment  of  (1)  add : 

Leucocyte  suspension  0.2  c.c. 

Salt  solution  0.3  c.c. 

To  the  leucocyte  sediment  of  (2)  add : 

Bacterial  suspension  0.1  c.c. 

Salt  solution  0.4  c.c. 

Allow  the  tubes  to  stand  for  30  minutes  at  37°  C.  Then  make 
smears  and  examine  as  in  Experiment  1. 

Materials: 

Serum,  leucocytes,  bacterial  Capillary   pipettes   with   rubber 

emulsion  as  described.  nipples. 

Microscope  slides.  Jenner's  stain. 

2  Centrifuge  tubes.  5  £-inch  test  tubes. 

LESSON  XXVII 
DETERMINATION  OF  OPSONIC  INDEX  (WRIGHT) 

PREPARE  the  following  reagents : 

1.  Bacillary     suspension.     Make     suspension    of    Staphylococcus 
aureus  culture  in  salt  solution.    Allow  to  stand  in  test  tube  for  clumps 
to  settle  out.    Transfer  upper  portion  to  separate  test  tube. 

2.  Leucocyte    suspension.     Obtain  15  or  more  drops  of  blood  from 
finger  tip  in  centrifuge  tube  containing  about  10  c.c.  of  citrate  solution. 


170  LABORATORY   COURSE   IN   SERUM   STUDY 

(Sodium  citrate  1  gram,  sodium  chloride  0.5  gram,  water  100  c.c.) 
Centrifugalize  and  wash  once  in  salt  solution.  With  capillary  pipette 
remove  the  upper  layer  of  the  sediment  which  will  contain  most  of  the 
leucocytes. 

3.  Serum.  Collect  about  10  drops  of  blood  in  Wright  capsule.1 
Seal  the  dry  end  in  the  flame  and  centrifugalize  to  obtain  serum. 

Use  portion  of  serum  to  make  control  pool,  mixing  serum  from  5 
to  10  different  individuals. 

TEST 

Use  capillary  pipette  l  with  mark  about  2  cm.  from  the  tip.  Take 
up  first  serum  to  the  mark,  then  air,  then  leucocytes  to  the  mark,  then 
bacteria.  Mix  the  reagents  in  a  watch  glass  and  again  draw  into  the 
pipette  and  seal  the  tip.  Incubate  one  half  hour.  Blow  out  contents 
of  capillary,  mix  thoroughly  and  make  smears.  Stain  with  Jenner's 
stain  4  minutes,  wash  and  blot. 

Each  student  makes  test  with  his  own  serum  and  with  a  control 
pool  which  does  not  include  his  own. 

The  opsonic  index  is  the  ratio  between  the  phagocytic  average 
of  the  specimen  with  the  patient's  serum  and  of  that  with  the 
normal  pool,  expressed  as  a  decimal,  i.e. 

"  Normal "  specimen  —  phagocyte  average  2.5 
Patient's  specimen  —  phagocyte  average    3.75 

Opsonic  index  1.5 

If  the  figures  for  normal  and  patient  were  reversed,  the  index 
would  be  0.66. 
Materials : 

Staphylococcus  suspension. 
Citrate  solution. 
Wright  capsules  and  pipettes. 
Jenner's  stain. 

LESSON  XXVIII 

DIFFERENCE  IN  HEAT  STABILITY  BETWEEN  NORMAL  AND 
IMMUNE   OPSONINS   OR  BACTERIOTROPINS 

PREPARE  materials  —  bacterial  (staphylococcus)  emulsion  — 
leucocytes  and  serum  as  for  opsonin  test  by  Wright's  method. 

1  See  Lesson  XIII,  page  88. 


172         LABORATORY  COURSE  IN  SERUM  STUDY 

Experiment  1 
HEAT  SUSCEPTIBILITY  OF  NORMAL  OPSONIN 

Divide  the  normal  serum  obtained  from  own  finger  into  two  parts. 
Heat  one  of  these  in  a  water  bath  at  56°  C.  for  30  minutes. 

Now  carry  out  opsonin  tests  in  parallel  (1)  with  unheated  serum, 
(2)  with  the  heated  serum  —  using  same  bacterial  emulsion  and  leuco- 
cytes in  both  cases. 

Compare  results  by  counting  slides. 

Experiment  2 
HEAT  SUSCEPTIBILITY  OF  IMMUNE  OPSONIN 

Staphylococcus  immune  rabbit  serum  will  be  given  out. 
Divide  into  two  parts.     Heat  one  part  to  56°  C.  for  20  minutes  in 
a  water  bath. 

Carry  out  parallel  tests  as  above  and  compare  the  results. 

Materials : 

Staphylococcus  emulsion. 

Glass  tubing  for  Wright  capsules  and  pipettes. 

Staphylococcus  immune  serum. 


LESSON  XXIX 
TITRATION  OF  NORMAL  OPSONIN  BY  DILUTION  METHOD 

TITRATE  opsonic  power  of  normal  guinea  pig  serum  for  Staphylo- 
coccus aureus  by  dilution  method  as  follows : 

1.  Make  dilutions  of  serum  1-5,  1-10,  1-20,  1-100. 

2.  Set  up  series  of  opsonic  tests  in  Wright  capillary  pipette,  using 
Staphylococcus  suspension,  suspension  of  guinea  pig  leucocytes  and 
diluted  serum  equal  parts.     Make  additional  test  with  undiluted  serum, 
and  also  control  with  leucocytes,  bacteria  and  salt  solution. 

3.  Incubate  30  minutes,  make  smears  and  stain  with  Jenner's  stain. 
Determine  by  counts  in  which  dilution  there  is  a  definite  increase  of 
phagocytosis  as  compared  with  the  salt  solution  control. 

Materials :  same  as  for  two  preceding  lessons. 


174         LABORATORY  COURSE  IN  SERUM  STUDY 

LESSON  XXX 
TITRATION  OF  IMMUNE  OPSONIN  BY  DILUTION  METHOD 

INTRODUCTORY  REMARKS 

THERE  are  several  methods  of  titrating  the  opsonic  power  of 
a  serum.  The  most  accurate  method  probably  is  that  in  which  a 
number  of  progressively  increasing  dilutions  of  the  serum  are 
made  and  the  opsonic  power  of  each  dilution  is  tested.  In  this 
way  the  degree  of  dilution  at  which  the  opsonic  power  of  the  serum 
disappears,  that  is,  at  which  there  is  not  more  phagocytosis  in 
the  specimen  with  this  serum  than  there  is  in  a  control  with  salt 
solution,  is  determined.  In  this  way  two  sera  can  be  compared 
as  to  their  opsonic  strength.  It  is  the  method  first  used  by  Klein 
in  the  case  of  typhoid  opsonins,  and  the  one  utilized  by  Jobling 
in  the  standardization  of  anti-meningococcus  serum. 

Reagents : 

1.  Suspension  of  Staphylococcus  aureus  from  24-hour  agar  slant. 

2.  Suspension  of  leucocytes  obtained  from  peritoneum  of  guinea 
pig  injected  with  aleuronat. 

(The  method  of  preparing  these  two  suspensions  has  been  given  in 
a  previous  lesson.) 

3.  Serum  of  rabbit  immunized  against  Staphylococcus  aureus. 
Before  beginning  the  experiment  0.2  c.c.  of  this  serum  should  be 

placed  in  test  tube  and  heated  one  half  hour  at  56°  for  Series  III  below. 

4.  Serum  of  normal  rabbit. 

Prepare  dilutions  of  each  of  the  sera,  1-5,  1-10,  1-20  and  1-100. 

I 

Set  up  a  series  of  six  tubes.  In  the  first  put  0.25  c.c.  of  undiluted 
immune  serum,  in  the  next  four  0.25  c.c.  of  1-5,  1-10,  1-20  and  1-100 
dilutions  respectively,  and  in  the  last  tube  0.25  c.c.  of  salt  solution  for 
control. 

II 

Set  up  a  similar  series  with  normal  serum,  omitting  the  fifth  and 
sixth  tubes. 


176  LABORATORY   COURSE   IN   SERUM    STUDY 

III 

Set  up  a  third  series  of  three  tubes  containing  immune  serum  which 
has  been  heated  at  56°  for  half  an  hour.  After  heating,  dilutions  of 
1-5,  1-10  and  1-20  should  be  prepared,  and  0.25  of  each  dilution  added 
to  one  of  the  tubes. 

To  each  of  these  tubes  now  add  0.1  c.c.  of  bacterial  suspension 
and  0.2  c.c.  of  washed  leucocytes.  Incubate  for  one  half  hour  and  pre- 
pare smears  from  the  sediment  of  each  tube.  Stain  the  smears  with 
Jenner's  stain  and  count  the  number  of  bacteria  contained  in  50  leuco- 
cytes in  each  smear,  calculating  the  average  number  per  leucocyte. 
Any  slide  which  shows  a  definite  increase  above  the  salt  solution  should 
be  regarded  as  showing  positive  opsonic  power  in  the  corresponding 
dilution  of  serum. 

Materials:  same  as  three  preceding  lessons. 

15  £-inch  t£st  tubes.  5  J-inch  test  tubes. 


LESSON  XXXI 
PREPARATION  OF  BACTERIAL  VACCINE 

THE  preparation  of  bacterial  vaccines  consists  in  four  steps : 

I.   PREPARATION  OF  THE  EMULSION 

Each  student  is  given  two  agar  slants  of  Staphylococcus  aureus  of 
24-hour  growth.  To  each  tube  add  about  2  c.c.  of  sterile  salt  solution 
with  a  sterile  pipette.  Remove  the  bacteria  from  the  surface  of  the  agar 
by  scraping  gently  with  a  sterile  platinum  loop,  being  careful  not  to  cut 
into  the  surface  of  the  agar.  When  an  even  suspension  is  obtained  this  is 
transferred  to  a  small  sterile  bottle  or  test  tube  containing  glass  beads 
and  thoroughly  shaken  to  break  up  clumps  of  the  organisms. 

II.   STANDARDIZATION 

The  second  step  in  the  preparation  of  vaccine  is  to  determine  the 
number  of  organisms  contained  per  c.c. 

(a)  Hcemocytometer  Method 

A  staining  solution  is  prepared  by  adding  to  20  c.c.  of  1  per  cent 
phenol  1  c.c.  of  a  saturated  alcoholic  solution  of  thionin.  A  small 


178          LABORATORY  COURSE  IN  SERUM  STUDY 

amount  of  the  carefully  shaken  bacterial  suspension  is  removed  to  a 
watch  glass.  A  1-100  dilution  is  prepared  in  a  red  cell  pipette  with  the 
staining  solution  as  diluent  to  the  101  mark.  After  carefully  shaking 
and  after  blowing  out  the  portion  of  the  fluid  in  the  capillary  end  of 
the  pipette,  a  small  drop  is  placed  in  a  counting  chamber  and  covered 
with  a  flat  cover  slip.  After  allowing  15  minutes  for  the  bacteria  to 
settle,  a  count  is  made,  with  a  No.  5  or  a  No.  6  lens,  of  a  number  of 
squares  until  200  or  more  bacteria  have  been  counted.  It  is  best  to 
take  this  count  from  different  portions  of  the  ruled  surface  and  from 
two  separate  drops  of  the  mixture.  The  small  squares  have  an  area 
of  ^rJ-fr  of  a  square  mm.,  the  depth  of  the  chamber  is  0.1  mm.,  the  dilu- 
tion is  1-100.  The  number  of  bacteria  may  be  estimated  by  the  fol- 
lowing formula : 

No.  of  bacteria  counted  X  400  X  10  X  100  X  1000  _  Dumber  of  bac- 

Number  of  squares  counted 

tend  in  i  c.c. 

(6)  Wright's  Method 

A  Wright  capillary  pipette  is  prepared  with  a  mark  about  one  inch 
from  the  tip.  A  small  puncture  is  made  in  the  tip  of  the  finger  and  a 
fresh  drop  of  blood  obtained.  Three  units  of  salt  solution  are  then 
drawn  up  in  the  pipette,  admitting  a  bubble  of  air  between  each  two 
portions  of  salt  solution.  Blood  from  the  finger  tip  is  then  drawn  up 
to  the  mark,  a  bubble  of  air  admitted,  and  bacterial  suspension  drawn 
up  to  the  mark.  The  mixture  is  then  blown  out  on  a  clean  slide  and 
drawn  in  and  out  of  the  pipette  several  times  to  insure  even  mixing  of 
the  blood  and  bacteria.  A  drop  of  this  mixture  is  placed  on  a  second 
slide  and  carefully  spread  across  the  slide  in  the  manner  of  making  blood 
smears.  It  is  important  that  the  film  be  thin  and  even  so  that  the  red 
cells  are  not  piled  in  masses  in  any  portion  of  the  film.  This  film  is 
stained  with  Jenner's  stain,  or  by  any  other  simple  method,  and  a  differ- 
ential count  of  the  number  of  bacteria  and  red  cells  in  a  number  of 
fields  in  different  parts  of  the  slide  is  made.  For  this  a  ruled  scale  to 
be  inserted  in  the  eyepiece  of  the  microscope  is  very  helpful.  A  num- 
ber of  fields  are  counted,  taken  at  random,  until  200  red  cells  have 
been  counted.  The  number  of  bacteria  in  the  suspension  may  then 
be  estimated  from  the  number  of  bacteria  counted,  using  the  following 
formula  (assuming  that  the  blood  of  the  worker  contains  5,000,000  red 
cells  per  cubic  mm.) : 

Number  of  bacteria  X  5,000,000  X  1000      XT 

Number  of  red  cells  (200)  "=  Number  °f  bactena  per  C'C' 


180          LABORATORY  COURSE  IN  SERUM  STUDY 

(c)  Other  Methods 

A  number  of  other  methods  have  been  devised  for  standardization 
of  vaccines. 

One  method  (Hopkins)  is  to  centrifugalize  at  high  speed  in  a  special 
tube  with  graduated  tip  until  the  supernatant  fluid  is  clear.  The  num- 
ber of  organisms  for  a  number  of  species  in  such  a  closely  packed 
sediment  has  been  determined,  and  is  as  follows : 

Staphylococcus  aureus  0.01  c.c.  equals  10  billion 

Streptococcus  haemolyticus  0.01  c.c.  equals    8  billion 

Gonococcus  0.01  c.c.  equals    8  billion 

Pneumococcus  (capsulated)  0.01  c.c.  equals  2.5  billion 

B.  typhosus  0.01  c.c.  equals    8  billion 

B.  coli  0.01  c.c.  equals    4  billion 

Another  method  of  standardization  is  by  the  turbidity  of  the  emul- 
sion. Standard  tubes  of  carefully  counted  suspensions  are  prepared, 
and  the  vaccine  to  be  standardized  is  then  diluted  until  the  turbidity 
is  equal  to  that  in  the  standard  tube. 

III.   STERILIZATION 

The  most  reliable  means  of  sterilization  is  by  means  of  heat.  For 
staphylococci  and  streptococci  59°  to  60°  C.  for  half  an  hour  is  satis- 
factory ;  for  typhoid  bacilli  55°  to  56°  for  an  hour  is  usually  used.  In 
heating,  the  bacteria  should  be  placed  in  a  sealed  tube  and  the  entire 
tube  immersed  in  the  water  bath.  After  exposure  to  heat  test  the  steril- 
ity of  the  vaccine  by  transferring  two  or  three  loopfuls  of  the  suspen- 
sion to  an  agar  slant. 

IV.   DILUTION 

The  stock  suspension  prepared  is  as  a  rule  too  concentrated  to  allow 
the  accurate  measurement  of  the  dose  desired,  which  may  vary  accord- 
ing to  the  organism  from  20  to  500  millions  or  even  more.  After 
the  vaccine  has  been  sterilized  by  heat,  sufficient  5  per  cent  phenol 
or  an  equivalent  quantity  of  other  aromatic  disinfectant  should  be 
added  to  bring  the  content  of  phenol  in  the  mixture  up  to  0.5  per  cent. 

Each  student  will  prepare  a  suspension  from  the  cultures 
given  out,  standardize  this  suspension  by  methods  (a)  and  (ft), 
heat  at  59°  for  half  an  hour,  test  for  sterility,  and  prepare  two 
dilutions,  one  containing  200  million  and  one  containing  1000 
million  per  c.c. 


182  LABORATORY   COURSE   IN   SERUM   STUDY 

Materials : 

2  agar  slants  of  staphylococcus  aureus. 
Sterile  saline  solution. 
Sterile  pipette. 

Sterile  tube  or  bottle  with  glass  beads. 
Haemocytometer. 

Thionin  solution  in  1  per  cent  phenol. 
Glass  slides. 
Wright  or  Jenner  stain. 
Phenol. 

LESSON  XXXII 
ANAPHYLAXIS 

INTRODUCTORY  REMARKS 

BECAUSE  of  the  large  number  of  animals  required  in  this  work, 
the  class  had  better  do  these  experiments  in  relatively  large 
groups. 

I.   ACTIVE  SENSITIZATION 

Four  guinea  pigs  of  about  200  grams  weight  are  etherized  on  operat- 
ing boards  for  intravenous  injection  into  external  jugular  veins. 

Guinea  pigs  (a)  and  (b)  receive  0.25  c.c.  horse  serum  diluted  with 
1.75  c.c.  salt  solution. 

Guinea  pigs  (c)  and  (d)  receive  0.25  c.c.  sheep  serum  diluted  with 
1.75  c.c.  salt  solution. 

This  should  be  done  two  weeks  before  the  time  for  Lesson  XXXII. 

Two  weeks  after  the  sensitizing  injection  the  pigs  are  again  injected 
intravenously  as  follows,  into  opposite  jugular  vein : 

(a)  0.5  c.c.  of  horse  serum. 
(6)  0.5  c.c.  of  sheep  serum, 
(c)  0.5  c.c.  of  sheep  serum. 

The  results  in  (a)  and  (c)  demonstrate  anaphylactic  shock. 
The  absence  of  symptoms  in  (b)  demonstrate  specificity. 

Guinea  pig  (d)  is  given  0.01  c.c.  sheep  serum  subcutaneously  four 
times,  at  one  hour  intervals.  Observe  carefully  symptoms  after  each 
injection.  Then  give  0.5  c.c.  sheep  serum  intravenously.  Symptoms 
will  be  slighter  than  in  (a)  and  (c).  This  demonstrates  desensitization. 


184  LABORATORY   COURSE   IN   SERUM   STUDY 

Materials  needed  for  each  group  of  students : 

4  guinea  pigs. 

1  c.c.  horse  serum. 

1  c.c.  sheep  serum. 

Syringe. 

Scalpel,  forceps,  scissors,  thread,  for  isolation  and  ligation  of  jugu- 
lar vein. 

Ether. 


LESSON  XXXIII 
ANAPHYLAXIS  (Continued) 

II.   PASSIVE  SENSITIZATION 

OBTAIN  serum  of  rabbit  which  has  been  highly  immunized  to  horse 
serum. 

Make  preliminary  titration  of  precipitin  of  this  serum  for  horse 
serum. 

Inject  1.0  c.c.  of  this  serum  intravenously  into  each  of  three  guinea 
pigs.  After  24  hours  inject  into  the  opposite  jugular  vein  as  follows  : 

Guinea  pig  (a)  0.1  c.c.  horse  serum 
Guinea  pig  (6)  0.3  c.c.  horse  serum 
Guinea  pig  (c)  1.0  c.c.  horse  serum 

Observe  symptoms. 

In  case  of  anaphylactic  death,  immediately  open  chest  and  observe 
condition  of  lungs  and  also  whether  heart  is  still  beating.  While  such 
an  animal  is  dying  it  is  important  to  watch  carefully  the  gradual  altera- 
1  tion  of  respirations,  the  great  irregularity  and  slowing  of  breathing  and 
the  intense  inspiratory  efforts  just  before  death. 


Printed  in  the  United  States  of  America. 


UNIVERSITY  OF  CALIFORNIA  UBRARY 


